Medicinal Composition of Extract of Seed of Emblica Officinalis and Method of Preparing the Same

ABSTRACT

A composition having an extract of seed of  Emblica officinalis . Methods of preparing extract of seed of  Emblica officinalis . An amla seed blend composition having various ratios of extracts of seeds of  Emblica officinalis . Nutraceutical or pharmaceutical methods for decreasing the total cholesterol, decreasing triglyceride, decreasing blood glucose level, enhancing HDL-C levels, increasing the HDL-C level to total cholesterol ratio, lowering LDL-C levels, decreasing the CRP level, decreasing the intima media thickening, reducing hair fall in mammals especially human beings. The extract of seed of  Emblica officinalis , or the amla seed blend composition is more effective compared to extracts prepared from fruits of  Emblica officinalis.

This Application is a divisional of U.S. patent application Ser. No.14/608,680 filed Jan. 29, 2015, which is a divisional of U.S. patentapplication Ser. No. 14/508,998, filed Oct. 7, 2014, which is acontinuation of International Patent Application No. PCT/1N2014/000642filed Oct. 7, 2014, which claims priority of Indian ProvisionalApplication Serial No. 4565/CHE/2013 filed Oct. 8, 2013.

FIELD

The disclosure relates a medicinal composition of the extract of seed ofEmblica officinalis, a method of preparing a composition consisting ofextract of seed of Emblica officinalis more particularly which hasapplication as a nutraceutical or pharmaceutical for reducing the totalcholesterol, reducing triglyceride, reducing blood glucose level,enhancing HDL-Cholesterol, increasing the HDL-Cholesterol to totalcholesterol ratio, lowering LDL-Cholesterol levels, reducing the CRPlevel, decreasing the intima media thickening, reducing hair fall inmammals especially human beings. The composition is effective even atlower dosage for reducing the total cholesterol, reducing triglyceride,reducing blood glucose level, enhancing HDL-Cholesterol levels,increasing the HDL-Cholesterol to total cholesterol ratio, loweringLDL-Cholesterol levels, reducing the CRP level, decreasing the intimamedia thickening and reducing hair fall.

BACKGROUND

Amla (or Amlaka, Amlaki, or other variants) is one of the mostfrequently used of the Ayurvedic herbs; it is the fruit of Phyllanthusemblica, also called Emblica officinalis. The fruit is similar inappearance to the common gooseberry (Ribes spp., a type of currant),which is botanically unrelated to amla. However, due to the similarappearance of the fruit clusters, amla is usually called the “Indiangooseberry.” The plant, a member of the Euphorbiaceae, grows to become amedium-sized tree that is found growing in the plains and sub-mountainregions all over the Indian subcontinent from 200 to nearly 2000 metersabove sea level. Indian gooseberry is a wonder herbs and one of theprecious gifts of nature to man. It contributes towards health andlongevity.

Emblica officinalis (EO) enjoys a hallowed position in Ayurveda— anIndian indigenous system of medicine. According to ancient Indianmythology, it is the first tree to be created in the universe. Emblicaofficinalis fruit is one of the key constituents of the celebratedAyurvedic preparation, Chyavanaprash, used in India for thousands ofyears as a vitalizing and rejuvenating health tonic. According toAyurveda, amla balances all three doshas. While amla is unusual in thatit contains five out of the six tastes recognized by Ayurved, it is mostimportant to recognize the effects of the “virya”, or potency, and“vipaka”, or post-digestive effect. The fruits of EO are widely used inthe Ayurveda and are believed to increase defense against diseases. I

Coronary heart disease (CHD) continues to be the major cause ofpremature death in most developed and developing countries. A low levelof HDL cholesterol is the second most important risk factor for CHD, asdemonstrated in numerous clinical and epidemiological studies (Gorden,D. and Rifkind, H. M., N. Engl. J. Med., 1989, 321:1311-1315; Brewer,Jr., H. B., New Engl. J. Med, 2004, 350:1491-94) and HDL has emerged,during the past decade, as a new potential target for the treatment ofcardiovascular diseases. The key role of HDL as a carrier of excesscellular cholesterol in the reverse cholesterol transport pathway isbelieved to provide protection against atherosclerosis. In reversecholesterol transport, peripheral tissues, for example, vessel-wallmacrophages, remove their excess cholesterol through the ATP-bindingcassette transporter 1 (ABCA1) to poorly lipidated apolipoprotein A-I,forming pre-.beta.-HDL. Lecithin-cholesterol acyltransferase thenesterifies free cholesterol to cholesteryl esters, converting pre-β-HDLto mature spherical α-HDL.

HDL cholesterol is transported to the liver by two pathways: 1) it isdelivered directly to the liver through interaction with the scavengerreceptor, class B, type I (SR-BI); 2) cholesteryl esters in HDL aretransferred by the cholesterol ester transferase protein (CETP) tovery-low-density-lipoproteins (VLDL) and low-density lipoproteins (LDL)and are then returned to the liver through the LDL receptor. HDLcholesterol that is taken up by the liver is then excreted in the formof bile acids and cholesterol, completing the process of reversecholesterol transport (Brewer, H. B. Jr., Arterioscl. Thromb. Vasc.Biol., 2004, 24:387-91). HDL is believed to have the ability to removecholesterol from macrophages, thus preventing the formation of foamcells.

A second beneficial role of HDL in atherosclerosis is in protecting LDLfrom oxidation (Navab, M. et al, Circulation, 2002, 105:290-92). Unlikenormal LDL, oxidized LDL is readily taken up by macrophage scavengerreceptor SR-A or CD36 resulting in the formation of foam cells. Foamcells are a major component of the early atherosclerotic lesion.Further, HDL may slow the progression of lesions by selectivelydecreasing the production of endothelial cell-adhesion molecules thatfacilitate the uptake of cells into the vessel wall (Barter, P. J., etal, Curr. Opin. Lipid, 2002, 13:285-88). HDL may also prolong thehalf-life of prostacycline and preserve its vasodilatory effect(Mackness, M. I. et al, Atherosclerosis, 1993, 104:129-35).

Several lines of evidence support the concept that increasing the HDLlevel may provide protection against the development of atherosclerosis.Epidemiologic studies have shown an inverse relation between HDLcholesterol levels and the risk of cardiovascular disease. Increasingthe HDL cholesterol level by 1 mg may reduce the risk of cardiovasculardisease by 2 to 3 per cent. Over expressing the apo-A-I gene intransgenic mice and rabbits and infusing complexes consisting of apo A-Iand phospholipids into hyperlipidemic rabbits increase HDL cholesterollevels and decrease the development of atherosclerosis (Brewer, H B,Jr., loc. cit). In humans, infusing either of these complexes orpro-apo-A-I results in short term increase in HDL cholesterol, biliarycholesterol and fecal cholesterol loss, reinforcing the concept thatelevating the HDL cholesterol level decreases the risk of cardiovasculardisease.

More than 40 per cent of patients with myocardial infarction have lowHDL-C as a cardiac risk factor. (Genest, J. J., et al, Am. J. Cardiol.,1991, 67:1185-89). In the prospective and multicentric EuropeanConcerted Action on Thrombosis and Disabilities (ECAT) Angina PectorisStudy, Bolibar et al (Thromb. Haemost., 2000, 84:955-61) identified lowHDL-C and low apoA-I as the most important biochemical risk factors forcoronary events in patients with angiographically assessed CHD. Byconvention, the risk threshold value of HDL-C has been defined as 35mg/dL (0.9 mmol/L) in men and 45 mg/dL (1.15 mmol/L) in women [Expertpanel on detection, evaluation and treatment of high blood cholesterolin adults. The second report of the National Cholesterol EducationProgram (NCEP) expert panel on detection, evaluation and treatment ofhigh blood cholesterol in adults (Adult Treatment Panel II).Circulation. 1994; 89:1329-1445)]. Because of interaction, the strengthof the association between HDL-C and cardiovascular risk depends on thepresence of additional risk factors. Therefore, threshold values arehigher in men with diabetes mellitus or hypercholesterolemia or in thepresence of multiple risk factors (von Eckardstein A, and Assmann G.Curr Opin Lipidol. 2000; 11:627-637). Low HDL-C has been identified asthe most frequent familial dyslipoproteinemia in patients with prematuremyocardial infarction (Genest, J. J. Jr., Circulation. 1992;85:2025-2033). Finally, in the Helsinki Heart Study (Manninen, V. et al,Circulation. 1992; 85:37-45) and the High-Density-LipoproteinCholesterol Intervention Trial of the Department of Veterans Affairs(VA-HIT) study (Rubins, H. B. et al, N Engl J Med. 1999; 341:410-418),increases of HDL-C on treatment with gemfibrozil were correlated withthe prevention of CHD events. Thus, HDL-C has become an importantcomponent of algorithms to assess the global cardiovascular risk ofpatients and also a target for therapeutic intervention and for thedefinition of treatment goals.

Strategies to correct dyslipidemia in atherosclerosis generally involvediet and/or drugs. The threshold serum total cholesterol and LDLcholesterol concentrations above which diet and drug therapy should beinitiated, as well as the goals of therapy, have been defined by theNational Cholesterol Education Program (JAMA, 1993, 269:3015-23). Thetarget serum LDL-C is <160 mg/dl (4.3 mmol/1) for patients with no riskfactors or only one risk factor for CHD; <130 mg/dl (3.4 mmol/1) forpatients with 2 or more risk factors and less than 100 mg/dl (2.6mmol/1) for those with CHD. Persons with diabetes also fall into thethird category. A reasonable target for triglyceride concentration is200 mg/dl or less; higher values are associated with a doubling of therisk of cardiovascular disease when serum cholesterol concentrationexceeds 240 mg/dl or when the LDL-C/HDL-C ratio exceeds 5:1.

A number of studies have shown that reducing serum LDL-C below thetarget levels does not necessarily result in proportional reduction inthe risk of CHD [(The Scandinavian Simvastatin Survival Study Group.Randomized trial of cholesterol lowering in 4444 patients with coronaryheart disease, Lancet, 1994, 344:1383-89; Shepherd, J. et al, N. Engl.J. Med., 1995, 333:1301-7; Sachs, F. M. et al, N. Engl. J. Med., 1998,315:1001-9; Circulation, 1998, 97:1446-52; The West of Scotland CoronaryPrevention Study Group, Circulation, 1998, 97:1440-45; Pederson, T. R.,Circulation, 1998, 97:1453-60] because of the attenuation of thecholesterol-heart disease relation at lower serum cholesterolconcentrations (Grundy, S. M., Circulation, 1998, 97:1436-39).

Dietary treatment of hyperlipidemia is a necessary foundation for drugtreatment. Depending on the degree of hyperlipidemia, the Step I andStep II diets can be introduced sequentially. The Step II diet containsno more than 30% of calories from fat, less than 7% of calories fromsaturated fatty acids and less than 200 mg of cholesterol per day. Inlong term studies, the Step II diet decreased serum LDL-C concentrations8-15% (Knopp, R. H., et al, JAMA, 1997, 278:1509-15; Walden, C. E.,Arterioscl. Thromb. Vasc. Biol., 1997, 17:375-82; Denke, M. A., Arch.Intern. Med., 1995, 156:17-26). Diets more restricted in fat than theStep II diet result in little additional reduction in LDL-C, raise serumTG concentration and lower HDL-C.

The point to note, from the above, is that reducing LDL-C alone is oflittle value in reducing the risk of CHD. Further, diets meant forreducing LDL-C may reduce HDL-C to a similar degree (Hunninghake, D. B.et al, N. Engl. J. Med., 1993, 328:1213-19; Schaefer, E. J., et al,Arterioscl. Thromb. Vasc. Biol., 1995, 15:1079-85); Stefanick, M. L., N.Engl. J Med, 1998, 339:12-20).

Drug therapy is resorted to when the desired effects are not achievedwith diets alone. Statins are the most popular among the lipid loweringdrugs. These drugs lower serum LDL-C concentrations by upregulatingLDL-receptor activity as well as reducing the entry of LDL into thecirculation. The maximal reductions achieved with a statin ranges from24-60%. Statins also reduce the serum TG levels; but they are ofteninsufficient. Statins are ineffective in the treatment of patients withchylomicronemia. Adverse effects of statins include, gastrointestinalupset, muscle aches and hepatitis. Rarer problems include myopathy(muscle pain with serum creatine kinase concentrations more than 1,000 Uper litre), rashes, peripheral neuropathy, insomnia, bad or vivid dreamsand difficulty in sleeping or concentrating (Abramowica, M., Med Lett.,1996, 38:67-70; Vgontzas, A. N. et al, Clin. Pharmacol. Ther., 1991,50:730-37; Roth, T. et al, Clin. Cardiol., 1992, 15:426-32; Partinen, M.et al, Am. J. Cardiol., 1994, 73:876-80). Other lipid-lowering drugsinclude bile acid-binding resins (e.g, cholesteramine and colestipol),nicotinic acid, and fibrates.

Drug therapy is not recommended for premenopausal women and men under 35years of age unless they have serum LDL-C concentrations of more than220 mg/dl (5.7 mmol/1), because their immediate risk of heart disease islow [Summary of the second report of the National Cholesterol EducationProgram (NCEP): expert panel on detection, evaluation and treatment ofhigh blood cholesterol in adults, JAMA, 1993, 269:3015-23].

Thus, diets alone or in conjunction with lipid lowering drugs fail toyield the desired goal of safe lipid lowering. However, this goal isachievable with the present inventive composition containing the activeprinciples of seed of Emblica officinalis. Emblica has been in safe usein India for thousands of years as component of Ayurvedic preparations.The composition from seed of Emblica officinalis offers the twinbenefits of reducing the harmful LDL cholesterol and enhancing thedesirable HDL cholesterol.

A number of studies have shown that Emblica officinalis is useful forreducing total cholesterol, reducing triglyceride, reducing LDLcholesterol and enhancing HDL cholesterol.

Ritu Mathur et al show the hypolipidaemic effect of fruit juice ofEmblica officinalis in cholesterol fed rabbits. The juice is obtainedfrom deseeded Emblica officinalis. U.S. Pat. No. 6,124,268, Ghosaldiscloses a natural antioxidant composition from Emblica officinalisusing pericarp of fresh berries (Emblica officinalis). Biswas Gopa et alshow the hypolipidemic efficacy of Amla (Emblica officinalis). The Amlaused is dried Amla fruit juice powder. Muhammed et al evaluated theanti-hyperglycemic and lipid-lowering properties of Emblica officinalispowder in normal and diabetic human volunteers. Zhang et al disclosesthe phenolic constituents of Emblica officinalis juice. Chatterjee et aldiscloses a novel compounds with hypocholesteremic activity from crudeEmblica officinalis (EO) fruit extracts. U.S. Pat. Nos. 7,780,996,8,158,167 and 8,455,020 discloses the method of reducing cholesterol,method of treating dyslipidemia and method of reducing triglyceride byextract of Emblica officinalis.

Amla is a fruit with wide range of medicinal properties. Our effort wasto find the most bioactive molecule/(s) or purified fraction havingbioactivity from Amla fruit. The fleshy part (pericarp) of Amla fruit isused for human consumption whereas Amla seeds are not edible anddiscarded. We evaluated different Amla extracts. Extracts prepared fromfresh fruit of Amla; fruit juice of whole Amla including the fleshy partand seeds of Amla; juice of fleshy part (pericarp); dried fruit; fleshof Amla fruit without seed; or Amla seed alone were evaluated for antihyperlipidemic activity. The methanol extract of all groups showedbeneficial activity, but the most unexpected and superior result wasobtained from the Amla seed alone extract. Amla seed alone extract wasable to significantly reduce the total cholesterol, LDL cholesterol,triglycerides, VLDL cholesterol and enhance the HDL cholestrol levels.Though Amla seed is not known to have any history of human consumption,we followed the lead with various extracts of Amla seed and found thatthe ethyl acetate portion of Amla seed extract is the most active. Theethyl acetate part was showing far superior activity compared to otherextracts with Amla seed and also against other extracts of Amla.

In view of the above, the disclosure provides a composition and methodof preparing an extract from the seed of Emblica officinalis unlikeother references where the extract is prepared from Emblica officinalis,especially from its fruits which found its application for the treatmentof reducing bad cholesterol, dyslipidemia and for reducing triglyceride.The disclosure provides a method of preparation of an extract of Emblicaofficinalis from the seed of Emblica officinalis and composition derivedcontain polyphenolic components and lipophilic components. The extractprepared from the seed of Emblica officinalis is useful for decreasingtotal cholesterol, decreasing triglyceride, reducing blood glucoselevel, enhancing HDL-Cholesterol level, increasing the HDL-Cholesterolto total cholesterol ratio, lowering LDL-Cholesterol level, reducing theCRP level, decreasing the intima media thickening even at a lower dosagelevel. The extract prepared from the seed of Emblica officinalis isuseful for reducing hair fall in humans by applying topically or by oraladministration.

SUMMARY

The disclosure provides a medicinal composition of the extracts of seedof Emblica officinalis (Amla seed extract). The Amla seed extractcomposition has applications as a nutraceutical or pharmaceuticalincluding for reducing the total cholesterol, reducing triglyceride,reducing blood glucose level, enhancing HDL-Cholesterol level,increasing the HDL-Cholesterol to total cholesterol ratio, loweringLDL-Cholesterol level, reducing the CRP level, decreasing the intimamedia thickening and reducing hair fall in mammals especially humanbeings.

The composition of the extracts of seed of Emblica officinalis issuperior compared to extract from fruits of Emblica officinalis forindications including decreasing total cholesterol, decreasingtriglyceride, decreasing blood glucose level, enhancing HDL-Cholesterollevel, increasing the HDL-Cholesterol to total cholesterol ratio,lowering LDL-Cholesterol levels, decreasing CRP level, decreasing theintima media thickening and decreasing hair fall.

When same dosages of Amla seed extract or Amla fruit extract wereadministered, Amla seed extract showed superior results compared to Amlafruit extract.

Even if the dosage of Amla fruit extract was increased compared to Amlaseed extract, Amla seed extract administration showed superior resultscompared to Amla fruit extract.

Some embodiments provide an amla seed blend composition (also referredto amla seed blend or Product 3). Product 3 is a blend of Product 1 andProduct 2. Product 1 includes alpha linolenic acid, linoleic acid andoleic acid. Product 2 includes triterpenoids and hydroxycinnamic acids.In some embodiments, Product 2 and Product 1 are blended in a ratio ofProduct 2 to Product 1 ranging from about 1:60 to about 99:1.

The disclosure provides a method of preparing extracts of seed ofEmblica officinalis. In some embodiments, the disclosed extracts of seedof Emblica officinalis can be used as a nutraceutical. In someembodiments, the disclosed extracts of seed of Emblica officinalis canbe used as a pharmaceutical. In some embodiments, administering thedisclosed extracts of seed of Emblica officinalis decreased the totalcholesterol level. In some embodiments, administering the disclosedextracts of seed of Emblica officinalis decreased triglyceride level. Insome embodiments, administering the disclosed extracts of seed ofEmblica officinalis decreased blood glucose level. In some embodiments,administering the disclosed extracts of seed of Emblica officinalisincreased level of HDL-Cholesterol. In some embodiments, administeringthe disclosed extracts of seed of Emblica officinalis increased theHDL-Cholesterol to total cholesterol ratio. In some embodiments,administering the disclosed extracts of seed of Emblica officinalislowered LDL-Cholesterol level. In some embodiments, administering thedisclosed extracts of seed of Emblica officinalis decreased the CRPlevel. In some embodiments, administering the disclosed extracts of seedof Emblica officinalis decreased thickening of the intima mediathickening. In some embodiments, administering the disclosed extracts ofseed of Emblica officinalis decreased hair fall.

In some embodiments, a method of producing the extract of seed ofEmblica officinalis is disclosed. The method includes selecting the rawmaterial (fresh fruit of Emblica officinalis), followed by deseeding thefruits of Emblica officinalis. Then the seeds of Emblica officinalis arecrushed and extracted with solvents. Solvents include methanol, ethanol,isopropanol, n-butanol, methyl acetate, ethyl acetate, propyl acetate,n-butyl acetate and combinations thereof to obtain mixture. The mixtureis filtered. The filtrate is concentrated to obtain a concentratedextract. The concentrated extract is dried to form a dried extract. Thedried extract is macerated with water and partitioned with ethylacetate. The ethyl acetate part and aqueous part are formed and collectthe ethyl acetate part. Ethyl acetate part is concentrated and dried toform powder of ethyl acetate extract of seed of Emblica officinalis.

The extract of seed of Emblica officinalis can be prepared from fresh ordried seed of Emblica officinalis.

The disclosure also provides a dosage form of the extract of seed ofEmblica officinalis. The disclosure provides a dosage form of an extractof seed of Emblica officinalis for oral administration. Dosage forms ofthe extract are selected from the group consisting of a capsule, tablet,granule, sachet, powder, paste, ointment, infusion, injection, ampoule,solution, suspension, emulsion, pills, oil, cream etc.

Further a dosage form of an extract of seed of Emblica officinalis isdisclosed for administering in a dosage ranging from about 5 mg to about500 mg to a human subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The above objectives and advantages of the disclosed teachings willbecome more apparent by describing in detail preferred embodimentsthereof with reference to the attached drawings in which:

FIG. 1: Flow chart depicting a method of preparation of ethyl acetateextract of methanol extract of seed of Emblica officinalis.

FIG. 2: Flow chart depicting a method of preparation of ethyl acetateextract of seed of Emblica officinalis.

FIG. 3: Flow chart depicting a method of preparation of pectinasetreated water extract of fruits of Emblica officinalis.

FIG. 4: Flow chart depicting a method of preparation of alcoholicextract of fruits of Emblica officinalis.

FIG. 5: Flow chart depicting a method of preparation of pectinasetreated water extract of fruits of Emblica officinalis without seed.

FIG. 6: Flow chart depicting a method of preparation of alcoholicextract of fruits of Emblica officinalis without seed.

FIG. 7: Flow chart depicting a method of preparation of powder of driedseed of Emblica officinalis.

FIG. 8: Flow chart depicting a method of preparation of powder of waterextract of dried seed of Emblica officinalis.

FIG. 9: Flow chart depicting a method of preparation of powder ofmethanol extract of dried seed of Emblica officinalis.

FIG. 10: Flow chart depicting a method of preparation of powder of fruitof Emblica officinalis.

FIG. 11: Flow chart depicting a method of preparation of powder of waterextract of fruit of Emblica officinalis.

FIG. 12: Flow chart depicting a method of preparation of powder ofmethanol extract of fruit of Emblica officinalis.

FIG. 13: Flow chart depicting a method of preparation of Amla seed blendcomposition.

DETAILED DESCRIPTION

The disclosure provides an extract of seed of Emblica officinalis. Thedisclosure also provides amla seed extract blend compositions.

The disclosure provides seed extracts of Emblica officinalis preparedfrom fresh or dry seeds of Emblica officinalis. In some embodiments, thedisclosed extracts of seed of Emblica officinalis can be used as anutraceutical. In some embodiments, the disclosed extracts of seed ofEmblica officinalis can be used as a pharmaceutical. In someembodiments, administering the disclosed extracts of seed of Emblicaofficinalis decreased the total cholesterol level. In some embodiments,administering the disclosed extracts of seed of Emblica officinalisdecreased triglyceride level. In some embodiments, administering thedisclosed extracts of seed of Emblica officinalis decreased bloodglucose level. In some embodiments, administering the disclosed extractsof seed of Emblica officinalis increased level of HDL-Cholesterol. Insome embodiments, administering the disclosed extracts of seed ofEmblica officinalis increased the HDL-Cholesterol to total cholesterolratio. In some embodiments, administering the disclosed extracts of seedof Emblica officinalis lowered LDL-Cholesterol level. In someembodiments, administering the disclosed extracts of seed of Emblicaofficinalis decreased the CRP level. In some embodiments, administeringthe disclosed extracts of seed of Emblica officinalis decreasedthickening of the intima media thickening. In some embodiments,administering the disclosed extracts of seed of Emblica officinalisdecreased hair fall.

In some embodiments, administering the disclosed extracts of seed ofEmblica officinalis promoted hair growth.

The composition of the extracts of seed of Emblica officinalis issuperior compared to extract from fruits of Emblica officinalis forreducing the total cholesterol, reducing triglyceride, reducing bloodglucose level, enhancing HDL-Cholesterol level, increasing theHDL-Cholesterol to total cholesterol ratio, lowering LDL-Cholesterollevels, reducing the CRP level, decreasing the intima media thickeningreducing hair fall and promoting hair growth.

When same dosages of Amla seed extract or Amla fruit extract wereadministered, Amla seed extract administration showed superior resultscompared to administering Amla fruit extract.

Even if the dosage of Amla fruit extract was increased compared to Amlaseed extract, Amla seed extract administration showed superior resultscompared to Amla fruit extract.

Some embodiments provide an extract of seeds of Emblica officinalis. Theextract of Emblica officinalis includes triterpenoids, hydroxycinnamicacids, fatty acids and polyphenols. The triterpenoids includes beta sitosterol, beta amyrin and lupeol. The hydroxycinnamic acids includeferulic acid and p coumaric acid. The fatty acids include alphalinolenic acid, linoleic acid, oleic acid, stearic acid and palmiticacid. In some embodiments, the extract prepared from seed of Emblicaofficinalis includes polyphenolic components and lipophilic components.

In some embodiments of the extract of seeds of Emblica officinalis,triterpenoids ranges from about 0.5 to about 20% of the extract. In someembodiments, the extract of seeds of Emblica officinalis has about 0.5%to about of 5% of hydroxy cinnamic acids. In some embodiments, theextract of seeds of Emblica officinalis has about 25% to about 50% offatty acids. In some embodiments, the extract of seeds of Emblicaofficinalis has about 10% to about 95% of polyphenols. In someembodiments, the extract of seeds of Emblica officinalis has about 10%to about 20% of polyphenols. Some embodiments provide an extract ofseeds of Emblica officinalis having about 0.5 to about 20%triterpenoids, about 25% to about 50% fatty acids and about 10% to about20% polyphenol. The triterpenoids include beta sito sterol, beta amyrinand lupeol, the hydroxycinnamic acids include ferulic acid andp-coumaric acid, the fatty acids include alpha linolenic acid, linoleicacid, oleic acid, stearic acid and palmitic acid, and about 2.5% toabout 50% of the polyphenols are hydroxycinnamic acids.

In some embodiments, the extract of seeds of Emblica officinalis hasabout 9.5% triterpenoids. In some embodiments, the extract of seeds ofEmblica officinalis has about 4.3% of hydroxycinnamic acids. In someembodiments, the extract of seeds of Emblica officinalis has about 41.8%fatty acids. In some embodiments, the extract of seeds of Emblicaofficinalis has about 15% polyphenols.

Some embodiments provide a seed extract product having the extract ofseed of Emblica officinalis. The seed extract product includes fillerssuch as lactose, spray dried lactose, starch, dibasic calcium phosphate,tribasic calcium phosphate, microcrystalline cellulose, hydroxy propylmethyl cellulose, or calcium carbonate.

The disclosure relates to a composition having polyphenolic andlipophilic components obtained from extract of seed of Emblicaofficinalis wherein the polyphenolic components present in the extractof seed of Emblica officinalis ranges from 10% to 95%. Similarly theextract of seed of Emblica officinalis contains lipophilic componentsranges from 5% and above.

Some embodiments provide a method of preparing an extract of seeds ofEmblica officinalis. The method includes deseeding fresh fruits ofEmblica officinalis to obtain seeds of Emblica officinalis. The seedsare crushed. The crushed seeds are extracted with 95% methanol to obtaina residue and a supernatant. The supernatant is concentrated to obtain aconcentrated methanol extract. The concentrated methanol extract isdried to obtain a powder of methanol extract of seeds of Emblicaofficinalis. The method further includes macerating the powder ofmethanol extract of seeds of Emblica officinalis in water to obtain aliquid. The liquid is extracted with ethyl acetate to obtain an ethylacetate phase. The ethyl acetate phase is concentrated to obtain aconcentrated ethyl acetate phase. The concentrated ethyl acetate phaseis dried to obtain a powder of ethyl acetate extract of methanol extractof seed of Emblica officinalis.

Some embodiments provide a method of preparing an extract of seeds ofEmblica officinalis. The method includes deseeding fresh fruits ofEmblica officinalis to obtain seeds of Emblica officinalis. The seedsare crushed. The crushed seeds are extracted with ethyl acetate toobtain a supernatant. The supernatant is concentrated to obtain aconcentrated ethyl acetate extract. The concentrated ethyl acetateextract is dried to obtain a powder of ethyl acetate extract of seeds ofEmblica officinalis.

In some embodiments, the fresh fruit of Emblica officinalis is cleanedand deseeded. Seeds are crushed and extracted for about 1 hr usingmethanol in an extractor with reflux condenser to obtain residue andsupernatant. The residue and supernatant is separated by draining outthe supernatant from the extractor bottom through the filter cloth. Theresultant supernatant is concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 65° C. to form concentratedextract. Later the concentrated extract is dried under vacuum at above500 mm of mercury to form powder of methanol extract of seed of EmblicaOfficinalis.

The powder of methanol extract of seed of Emblica officinalis ismacerated with water and partitioned with ethyl acetate. Collect theethyl acetate part. Concentrate the ethyl acetate part in an Agitatedthin film evaporator and dried under vacuum at above 500 mm of mercuryto form powder of ethyl acetate extract of methanol extract of seed ofEmblica officinalis. [FIG. 1]

In one embodiment fresh fruit of Emblica officinalis is cleaned anddeseeded. Seeds are crushed and extracted for 5 hrs using ethyl acetateat 78° C. in a Soxhlet extractor and then filtered. The resultantextract is concentrated in an Agitated thin film evaporator (ATFE) at atemperature of 75° C. to form concentrated extract. Later theconcentrated extract is dried under vacuum at above 500 mm of mercury toform powder of ethyl acetate extract of seed of Emblica officinalis.[FIG. 2]

In another embodiment the method of manufacture of a powder of apectinase treated water extract of fruits of Emblica officinalis is bypulping fruits of Emblica officinalis with demineralized water to createslurry. The slurry is treated with pectinase and then filtered to obtaina solution. The solution is concentrated and dried under vacuum. Driedproduct is pulverized and sieved through 30 mesh to obtain a powder ofthe pectinase treated water extract of fruits of Emblica officinalis.[FIG. 3]

Some embodiments disclose a method of preparing a powder of an alcoholicextract of fruits of Emblica officinalis. Fresh fruits of Emblicaofficinalis are pulverized and extracted for about 1 hr using 95%methanol in an extractor with reflux condenser to obtain residue andsupernatant. The residue and supernatant is separated by draining outthe supernatant from the extractor bottom through the filter cloth. Theresultant supernatant is concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 65° C. to form concentratedextract. Later the concentrated extract is dried under vacuum at above500 mm of mercury to form powder of methanol extract of fruit of EmblicaOfficinalis. [FIG. 4]

In some embodiments, a method of manufacture of a powder of a pectinasetreated water extract of fruits of deseeded Emblica officinalis isdisclosed. Fruit of deseeded Emblica officinalis is made into a pulpalong with demineralized water to create slurry. The slurry is treatedwith pectinase and then filtered to obtain a solution. The solution isconcentrated and dried under vacuum. The dried material is pulverizedand sieved through 30 mesh to obtain a powder of the pectinase treatedwater extract of fruits of deseeded Emblica officinalis. [FIG. 5]

In another embodiment, a method of preparing a powder of an alcoholicextract of fruits of deseeded Emblica officinalis is disclosed. Freshfruits of Emblica officinalis are deseeded and deseeded fruits arepulverized and extracted for about 1 hr using 95% methanol in anextractor with reflux condenser to obtain residue and supernatant. Theresidue and supernatant is separated by draining out the supernatantfrom the extractor bottom through the filter cloth. The resultantsupernatant is concentrated in an Agitated thin film evaporator (ATFE)at a temperature of 65° C. to form concentrated extract. Later theconcentrated extract is dried under vacuum at above 500 mm of mercury toform powder of methanol extract of fruit of deseeded EmblicaOfficinalis. [FIG. 6]

Some embodiments provide a composition having the extract of seeds ofEmblica officinalis. Some embodiments provide, a method of treatment byadministering to a human subject about 5 mg to about 500 mg of theextract of seeds of Emblica officinalis Some embodiments provide adosage form having the extract of seeds of Emblica officinalis. Thedosage form includes a dosage of the extract of seeds of Emblicaofficinalis ranging from about 5 mg to about 500 mg. Some embodiments ofthe method administer a dose of about 5 mg to about 500 mg per day to ahuman. Some embodiments administer a dose of about 5 mg to about 500 mgtwo or three times per day to a human. In some embodiments, the extractof seed of Emblica officinalis is administered in a dosage of 5 mg to100 mg in humans. The dosage form is administered in single or multipledoses per day. Some embodiments provide a dosage form such as a capsule,tablet, granule, sachet, powder, paste, ointment, infusion, injection,ampoule, solution, suspension, emulsion, pills, oil, or, cream.

Some embodiments provide a method of reducing total cholesterol byadministering an extract of seeds of Emblica officinalis. Someembodiments provide a method of reducing triglyceride by administeringan extract of seeds of Emblica officinalis. Some embodiments provide amethod of reducing blood glucose level by administering an extract ofseeds of Emblica officinalis. Some embodiments provide a method ofenhancing HDL-Cholesterol by administering an extract of seeds ofEmblica officinalis. Some embodiments provide a method of increasing aratio of HDL cholesterol to total cholesterol by administering anextract of seeds of Emblica officinalis. Some embodiments provide amethod of lowering LDL-Cholesterol levels by administering an extract ofseeds of Emblica officinalis. Some embodiments provide a method oflowering VLDL by administering an extract of seeds of Emblicaofficinalis. Some embodiments provide a method of reducing CRP level byadministering an extract of seeds of Emblica officinalis. Someembodiments provide a method of decreasing the intima media thickeningby administering an extract of seeds of Emblica officinalis. Someembodiments provide a method of reducing hair fall by administering anextract of seeds of Emblica officinalis.

Some embodiments provide an amla seed blend composition. Someembodiments of the amla seed blend composition are referred to asProduct 3. The amla seed blend composition is a blend of varying ratiosof Product 1 and Product 2. Product 1 includes alpha linolenic acid,linoleic acid and oleic acid. Product 2 includes triterpenoids andhydroxycinnamic acids. Product 2 includes triterpenoids and polyphenols.In some embodiments, Product 2 and Product 1 are blended in a ratio ofProduct 2 to Product 1 ranging from about 1:1 to about 99:1. In someembodiments, Product 2 and Product 1 are blended in a ratio of Product 2to Product 1 ranging from about 1:60 to about 99:1. In some embodiments,Product 2 and Product 1 are blended in a ratio of Product 2 to Product 1ranging from about 1:1 to about 1:10. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 2:3.In some embodiments, Product 2 and Product 1 are blended in a ratio ofProduct 2 to Product 1 of about 1:2. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 1:1.In some embodiments, Product 2 and Product 1 are blended in a ratio ofProduct 2 to Product 1 of about 3:2. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 10:1or 90:9. In some embodiments, Product 2 and Product 1 are blended in aratio of Product 2 to Product 1 of about 95:5 or 19:1. In someembodiments, Product 2 and Product 1 are blended in a ratio of Product 2to Product 1 of about 3:1 or 75:25. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 1:5.In some embodiments, Product 2 and Product 1 are blended in a ratio ofProduct 2 to Product 1 of about 1:10. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 1:3.

In some embodiments, the blending of Product 2 and Product 1 provides anamla seed blend composition having about 6% to about 50% oftriterpenoids. The triterpenoids include among others beta-sitosterol,beta amyrin and lupeol. In some embodiments, the blending of Product 2and Product 1 provides an amla seed product having about 2% to about 20%of hydroxycinnamic acids. The hydroxycinnamic acids include ferulic acidand p-coumaric acid. In some embodiments, combining Product 2 andProduct 1 results in an amla seed product having about 10% to about 60%of fatty acids. The fatty acids include unsaturated and saturated fattyacids. The unsaturated fatty acids includes alpha linolenic acid,linoleic acid and oleic acid.

Some embodiments provide a method of preparing an amla seed blend havingProduct 1 and the Product 2. Some embodiments provide a method ofpreparing the Product 1. Fresh fruits of Emblica officinalis aredeseeded to obtain seeds of Emblica officinalis. The seeds are crushed.The crushed seeds are extracted with 95% methanol to obtain a residueand a supernatant. The supernatant is concentrated resulting in aconcentrated methanol extract. The concentrated methanol extract isdried to obtain a powder of methanol extract of seeds of Emblicaofficinalis. The powder of methanol extract of seeds of Emblicaofficinalis is dispersed in water to obtain a dispersion. The dispersionis extracted with hexane in a liquid-liquid extractor following which ahexane and a water phase is collected. The hexane phase is concentratedto obtain a liquid form of a concentrated hexane extract. The liquidform of the concentrated hexane extract is cooled, whereby obtainprecipitates or crystals are formed and a liquid portion is obtained.The liquid portion is separated from the precipitates or crystals toobtain a liquid Product 1. Some embodiments provide a method ofpreparing Product 2 from Amla seed. The method includes extracting thewater phase collected above after hexane extraction with ethyl acetateto obtain an ethyl acetate phase. The ethyl acetate phase isconcentrated to obtain a concentrated ethyl acetate phase. Theconcentrated ethyl acetate phase is dried to obtain a powder of an ethylacetate extract. The powder of ethyl acetate extract is mixed with waterto obtain a liquid of powder of ethyl acetate extract. The liquid ofpowder of ethyl acetate extract is loaded onto an ion exchange column.The ion exchange column is eluted with water to obtain a water fraction(also referred to as Fraction 1). Next, the ion exchange column iseluted with 50% methanol to obtain a Fraction 2. Then the ion exchangecolumn is eluted with 80% methanol to obtain a Fraction 3. Fraction 1 isconcentrated to obtain a concentrate of Fraction 1. The concentrate ofFraction 1 is dried to obtain a powder of Fraction 1. Fraction 2 isconcentrated to obtain a concentrate of Fraction 2. The concentrate ofFraction 2 is dried to obtain a powder of Fraction 2. Fraction 3 isconcentrated to obtain a concentrate of Fraction 3. The concentrate ofFraction 3 is dried to obtain a powder of Fraction 3. The powder ofFraction 1, the powder of Fraction 2 and the powder of Fraction 3 arecombined to obtain Product 2. Some embodiments of Product 2 have thepowder of Fraction 1, the powder of Fraction 2 and the powder ofFraction 3 in a 0.5:1:0.75 ratio of Fraction1:Fraction 2:Fraction 3.Product 1 includes alpha linolenic acid, linoleic acid and oleic acid.Product 2 includes triterpenoids and hydroxycinnamic acids. In someembodiments, Product 2 and Product 1 are blended in a ratio of Product 2to Product 1 ranging from about 1:1 to about 99:1. In some embodiments,Product 2 and Product 1 are blended in a ratio of Product 2 to Product 1ranging from about 1:60 to about 99:1. In some embodiments, Product 2and Product 1 are blended in a ratio of Product 2 to Product 1 rangingfrom about 10:1 to about 1:10. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 2:3.In some embodiments, Product 2 and Product 1 are blended in a ratio ofProduct 2 to Product 1 of about 1:2. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 1:1.In some embodiments, Product 2 and Product 1 are blended in a ratio ofProduct 2 to Product 1 of about 3:2. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 10:1or 90:9. In some embodiments, Product 2 and Product 1 are blended in aratio of Product 2 to Product 1 of about 95:5 or 19:1. In someembodiments, Product 2 and Product 1 are blended in a ratio of Product 2to Product 1 of about 3:1 or 75:25. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 1:5.In some embodiments, Product 2 and Product 1 are blended in a ratio ofProduct 2 to Product 1 of about 1:10. In some embodiments, Product 2 andProduct 1 are blended in a ratio of Product 2 to Product 1 of about 1:3.[FIG. 13]

Some embodiments provide a dosage form having the amla seed blendcomposition, which is a blend of Product 1 and Product 2. The dosageforms include a dosage of the amla seed blend composition ranging fromabout 5 mg to about 500 mg per dose in human subject. The dosage form isadministered in single or multiple doses per day. Some embodimentsprovide a dosage form such as a capsule, tablet, granule, sachet,powder, paste, ointment, infusion, injection, ampoule, solution,suspension, emulsion, pills, oil, or, cream.

Some embodiments provide an amla seed blend composition, which is ablend of Product 1 and Product 2. Some embodiments provide a method ofreducing total cholesterol by administering the amla seed blendcomposition. Some embodiments provide a method of reducing triglycerideby administering the amla seed blend composition. Some embodimentsprovide a method of reducing blood glucose level by administering anamla seed blend composition. Some embodiments provide a method ofenhancing HDL-Cholesterol by administering an amla seed blendcomposition. Some embodiments provide a method of increasing a ratio ofHDL cholesterol to total cholesterol by administering an amla seed blendcomposition. Some embodiments provide a method of loweringLDL-Cholesterol levels by administering an amla seed blend composition.Some embodiments provide a method of lowering VLDL by administering anamla seed blend composition. Some embodiments provide a method ofreducing CRP level by administering an amla seed blend composition. Someembodiments provide a method of decreasing the intima media thickeningby administering an amla seed blend composition. Some embodimentsprovide a method of reducing hair fall by administering an amla seedblend composition.

It will be readily understood by the skilled artisan that numerousalterations may be made to the examples and instructions given herein.These and other objects and features of the invention will be madeapparent from the following examples. The following examples asdescribed are not intended to be construed as limiting the scope of thepresent invention.

Example-1 Method of Preparation of Ethyl Acetate Extract of MethanolExtract of Seed of Emblica officinalis (Amla Seed Extract)

Fresh fruits of Emblica officinalis (Amla) were collected (500 Kg).Fruits were deseeded by deseeding machine and fresh seeds (75 Kg) werecrushed through roller mill. 95% Methanol in an amount 2 times thequantity of crushed seeds was added to the crushed seeds to form amixture for methanol extraction. The extraction was performed using anextractor with reflux condenser. The bottom of the extractor was fittedwith a polypropylene (100 microns) filter cloth. The mixture wasrefluxed for one hour at 65° C. to obtain a first residue andsupernatant. The residue and supernatants were separated by draining outthe supernatant from the extractor bottom through the polypropylenefilter cloth using a centrifugal pump. After the first extraction, thefirst residue was further extracted with two times the quantity ofmethanol at 65° C. to get second residue and supernatant. The secondresidue was further extracted with two times the quantity of methanol at65° C. to get third residue and supernatant. All the supernatants werepooled and concentrated in an Agitated thin film evaporator (ATFE) at atemperature of 65° C. to form concentrated methanol extract.Concentrated methanol extract was dried under vacuum at above 500 mm ofmercury to obtain 5 kg of powder of methanol extract of seed of EmblicaOfficinalis.

The powder of methanol extract of seed of Emblica officinalis wasmacerated with water and transferred into a liquid-liquid extractor andextracted with ethyl acetate. Ethyl acetate phase and aqueous phase wereseparated. After extraction ethyl acetate phase was collected. Ethylacetate phase was concentrated in an Agitated thin film evaporator toform concentrated ethyl acetate extract. Ethyl acetate concentrate wasfed into vacuum stripper and dried under vacuum at above 500 mm ofmercury to obtain 2.5 kg of powder of ethyl acetate extract of methanolextract of seed of Emblica Officinalis. [FIG. 1]

Example-2 Method of Preparation of Ethyl Acetate Extract of Seed ofEmblica officinalis (Amla Seed Extract)

Fresh fruits of Emblica officinalis were collected (500 Kg). Fruits weredeseeded by deseeding machine and fresh seeds (75 Kg) were crushedthrough roller mill. Crushed seeds were filled in the Soxhlet extractorand extracted with ethyl acetate (300 L). The extraction was carried outfor 5 hrs at a temperature of about 78° C. After the completion ofextraction, the supernatant was filtered and concentrated in an Agitatedthin film evaporator (ATFE) at a temperature of 75° C. to formconcentrated ethyl acetate extract. Concentrated ethyl acetate extractwas dried under vacuum at above 500 mm of mercury to get 2 Kg of powderof ethyl acetate extract of seed of Emblica Officinalis. [FIG. 2]

Example-3 Method of Preparation of Pectinase Treated Water Extract ofFruits of Emblica officinalis (Amla)

Fresh fruits of Emblica officinalis were collected (100 Kg). Freshfruits of Emblica officinalis were pulped with demineralized water tocreate slurry. The slurry was treated with pectinase and then filteredto obtain a solution. The solution was concentrated and dried undervacuum. Dried product (5 kg) was pulverized and sieved through 30 meshesto obtain a powder of a pectinase treated water extract of Emblicaofficinalis. [FIG. 3]

Example-4

Method of preparation of alcoholic extract of fruits of Emblicaofficinalis (Amla) Fresh fruits of Emblica officinalis were collected(100 Kg). Fresh fruits of Emblica officinalis were pulverized. 95%Methanol in an amount 2 times the quantity (200 L) of pulverized fruitswere added to form a mixture for methanol extraction. The extraction wasperformed using an extractor with reflux condenser. The bottom of theextractor was fitted with a polypropylene (100 microns) filter cloth.The mixture was refluxed for one hour at 65° C. to obtain a firstresidue and supernatant. The residue and supernatants were separated bydraining out the supernatant from the extractor bottom through thepolypropylene filter cloth using a centrifugal pump. After the firstextraction, the first residue was further extracted with two times thequantity of methanol at 65° C. to get second residue and supernatant.The second residue was further extracted with two times the quantity ofmethanol at 65° C. to get third residue and supernatant. All thesupernatants were pooled and concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 65° C. to form concentratedmethanol extract. Concentrated methanol extract was dried under vacuumat above 500 mm of mercury to obtain 5 kilograms of powder of methanolextract of fruit of Emblica Officinalis. [FIG. 4]

Example-5 Method of preparation of pectinase treated water extract ofdeseeded Emblica officinalis (Amla)

Fresh fruits of Emblica officinalis were collected (100 Kg). Freshfruits of Emblica officinalis were deseeded by deseeding machine.Deseeded Emblica officinalis fruits (85 Kg) were pulped withdemineralized water to create slurry. The slurry was treated withpectinase and then filtered to obtain a solution. The solution wasconcentrated and dried under vacuum. Dried product (4 kg) was pulverizedand sieved through 30 meshes to obtain a powder of a pectinase treatedwater extract of deseeded Emblica officinalis. [FIG. 5]

Example-6 Method of Preparation of Alcoholic Extract of Deseeded Emblicaofficinalis (Amla)

Fresh fruits of Emblica officinalis were collected (100 Kg). Freshfruits of Emblica officinalis were deseeded by deseeding machine.Deseeded Emblica officinalis fruits (85 Kg) were pulverized. 95%methanol in an amount 2 times the quantity (170 L) of pulverized fruitswas added to form a mixture for methanol extraction. The extraction wasperformed using an extractor with reflux condenser. The bottom of theextractor was fitted with a polypropylene (100 microns) filter cloth.The mixture was refluxed for one hour at 65° C. to obtain a firstresidue and supernatant. The residue and supernatants were separated bydraining out the supernatant from the extractor bottom through thepolypropylene filter cloth using a centrifugal pump. After the firstextraction, the first residue was further extracted with two times thequantity of methanol at 65° C. to get second residue and supernatant.The second residue was further extracted with two times the quantity ofmethanol at 65° C. to get third residue and supernatant. All thesupernatants were pooled and concentrated in an Agitated thin filmevaporator (ATFE) at a temperature of 65° C. to form concentratedmethanol extract. Concentrated methanol extract was dried under vacuumat above 500 mm of mercury to obtain 3.5 kilograms of powder of methanolextract of deseeded fruit of Emblica Officinalis. [FIG. 6]

Example 7 Method of Preparation of Powder of Dried Amla Seed

Fresh fruits of Emblica officinalis (Amla) were collected (10 Kg).Fruits were deseeded by deseeding machine and fresh seeds (1.5 Kg) weredried in tray drier at 40° C. The dried material was powdered to obtaina powder of dried seed of Emblica officinalis (0.75 kg). [FIG. 7]

Example 8 Method of Preparation of Water Extract of Dried Amla Seed

Fresh fruits of Emblica officinalis (Amla) were collected (500 Kg).Fruits were deseeded by deseeding machine and fresh seeds (75 Kg) weredried in tray drier at 40° C. The dried seeds were crushed and chargedin to an extractor. Around 200 Litres of water was added into thecrushed seed and kept for a contact time of 3 hrs. Then the water partwas collected and fresh water was again added into seeds and repeatedthe extraction thrice. All the water parts were pooled, filtered andconcentrated in an evaporator, when the concentrated water extract ofdried seed reached the bottom of the vessel, the concentrate was fedinto drier and dried under vacuum above 500 mm of mercury. Dried productwas discharged from the bottom of the vessel and pulverized to obtain apowder of the water extract of dried seed of Emblica officinalis (4 Kg).[FIG. 8]

Example 9 Method of Preparation of Methanol Extract of Dried Amla Seed

Fresh fruits of Emblica officinalis (Amla) were collected (500 Kg).Fruits were deseeded by deseeding machine and fresh seeds (75 Kg) weredried in tray drier at 40° C. The dried seeds were crushed and chargedinto an extractor. Around 200 litres of 95% methyl alcohol was pumpedinto the extractor and kept for a contact time of 3 hours. Then thesolvent part (methanol part) was collected and fresh methyl alcoholpumped again into the extractor and extraction repeated thrice. All theextracts (methanol part) were pooled, filtered and dried in an Agitatedthin film drier (ATFD) which was working under vacuum 700 mm Mercury.Dried product was discharged from the bottom of the vessel and thenpulverized to obtain of powder of an alcoholic extract of seed ofEmblica officinalis (5 Kg). [FIG. 9]

Example 10 Method of Preparation of Powder of Dried Amla Fruit

Fresh fruits of Emblica officinalis 100 kgs were washed and chopped intoflakes and dried in a hot air oven at around 110° C. for 10 hours. Thedried material (9 kg) was powdered to obtain a powder of dried fruits ofEmblica officinalis. [FIG.: 10]

Example 11 Method of Preparation of Water Extract of Dried Amla Fruit

Fresh fruits of Emblica officinalis were collected (100 kg). Freshfruits were washed and chopped into flakes and dried in a hot air ovenat around 110° C. for 10 hours. Dried flakes were charged in to anextractor and around 200 Liters of water was added into the dried flakesand kept for a contact time of 3 hrs. Then the water part was collectedand water was again added into flakes and repeated thrice. All the waterparts were pooled, filtered and concentrated in an evaporator, when theconcentrated water extract of dried fruit reached the bottom of thevessel, the concentrate was fed into drier and dried under vacuum above500 mm of mercury. Dried product (6 kg) was discharged from the bottomof the vessel and pulverized to obtain a powder of the water extract ofdried fruits of Emblica officinalis. [FIG. 11]

Example 12 Method of Preparation of Methanol Extract of Dried Amla Fruit

Fresh fruits of Emblica officinalis were collected (100 kg). Freshfruits were washed and chopped into flakes and dried in a hot air ovenat around 110° C. for 10 hours. Dried flakes were charged in to anextractor and around 200 liters of 95% methyl alcohol was pumped intothe extractor and kept for a contact time of 3 hours. Then the solventpart (methanol part) was collected and fresh methyl alcohol pumped againinto the extractor and extraction repeated thrice. All the extracts(methanol part) were pooled, filtered and dried in an Agitated thin filmdrier (ATFD) which was working under vacuum 700 mm Mercury. Driedproduct (5 Kg) was discharged from the bottom of the vessel and thenpulverized to obtain of powder of an alcoholic extract of fruits ofEmblica officinalis. [FIG. 12]

Example 13

Fresh fruits of Emblica officinalis (Amla) were collected (500 Kg).Fruits were deseeded by deseeding machine to obtain fresh seeds of Amla.The fresh seeds (75 Kg) were crushed through roller mill. 95% Methanolin an amount 2 times the quantity of crushed seeds was added to thecrushed seeds to form a mixture for methanol extraction. The extractionwas performed using an extractor with reflux condenser. The bottom ofthe extractor was fitted with a polypropylene (100 microns) filtercloth. The mixture was refluxed for one hour at 65° C. to obtain a firstresidue and supernatant. The first residue and supernatant wereseparated by draining out the supernatant from the extractor bottomthrough the polypropylene filter cloth using a centrifugal pump. Afterthe first extraction, the first residue was further extracted with twotimes the quantity of methanol at 65° C. to get a second residue andsupernatant. The second residue was further extracted with two times thequantity of methanol at 65° C. to get a third residue and supernatant.All the supernatants were pooled and concentrated in an Agitated thinfilm evaporator (ATFE) at a temperature of 65° C. to form a concentratedmethanol extract. The concentrated methanol extract was dried undervacuum at above 500 mm of mercury to obtain 5 kg of powder of methanolextract of seed of Emblica Officinalis.

The powder of methanol extract of seed of Emblica officinalis wasdispersed in water and transferred into a liquid-liquid extractor andextracted with hexane. After extraction hexane phase and aqueous phaseseparated. Then the hexane phase was collected through side valve.Hexane phase was concentrated in an Agitated thin film evaporator toform concentrated hexane extract (2 Kg). The concentrated hexane extractwas cooled at 4° C. and kept cold for 24 hrs. Some components of theconcentrated hexane extract precipitated or crystallized by thiscooling. The crystals or precipitates were separated from the liquidcooled concentrated hexane extract by passing the cooled concentratedhexane extract through a filter press. The precipitates or crystals werefound to contain high melting point components such as palmitic acid andstearic acid. 1.5 kilograms of liquid (Product 1) was obtained afterpassing through the cooled concentrated hexane extract in the filterpress. Product 1 was found to contain unsaturated fatty acids such asalpha-linolenic acid, linolenic acid, and oleic acid.

Ethyl acetate was added to aqueous phase in a liquid-liquid extractorfor extraction. After extraction ethyl acetate phase and aqueous phasewere separated and ethyl acetate phase was collected through side valve.Ethyl acetate phase was concentrated in an Agitated thin film evaporatorto form concentrated ethyl acetate extract. Ethyl acetate concentratewas fed into vacuum stripper and dried under vacuum at above 500 mm ofmercury to obtain 2.5 kilograms of powder of ethyl acetate extract ofmethanol extract of seed of Emblica Officinalis. This extract was foundto contain triterpenoids and polyphenols. Polyphenols includedhydroxycinnamic acid.

Powder of ethyl acetate extract of methanol extract of seed of Emblicaofficinalis was mixed with water and loaded in a column having a FPX 66ion-exchange resin (Rohm &Haas, Philadelphia, USA). The columnchromatography was performed to further purify the triterpenoids, and,to separate hydroxycinnamic acids from other polyphenols. Column wasinitially eluted with water and water fraction was collected. Then thecolumn was eluted with different concentrations of methanol (50%methanol and 80% methanol) and collected the different methanolfractions. Water fraction was concentrated and dried under vacuum toform powder of water elute of ethyl acetate extract of seed of Emblicaofficinalis (0.1 kg) (Fraction 1). Fraction 1 was found to contain smallamount of triterpenoids which was confirmed by HPLC. The 50% methanolfraction was concentrated and dried under vacuum to form powder of 50%methanol elute of ethyl acetate extract of seed of Emblica officinalis(1 kg) (Fraction 2). Fraction 2 was found to contain triterpenoids andit was confirmed by HPLC.

80% methanol fraction was concentrated and dried under vacuum to formpowder of 80% methanol elute of ethyl acetate extract of seed of Emblicaofficinalis (0.75 kg) (Fraction 3). Fraction 3 was found to containhydroxycinnamic acids confirmed by HPLC method.

Fractions 1, 2 and 3 were combined to form a Product 2. Product 2 wasblended with Product 1 in a 2:3 ratio to form an amla seed blend Product3 (3.35 Kg). [FIG. 13] Product 3 had about 20% triterpenoids, about11.7% hydroxycinnamic acids, about 46.2% unsaturated fatty acids andabout 0.3% saturated fatty acids. Product 3 included about 9% beta sitosterol, about 6% beta amyrin, about 5% lupeol, about 7.5% ferulic acid,about 4.2% coumaric acid, about 20.5% alpha linolenic acid, about 15.8%linolenic acid, about 9.9% oleic acid, about 0.2% stearic acid and about0.1% palmitic acid.

Example 14 Analysis of Triterpenoids by HPLC

The triterpenoids was estimated by high performance liquidchromatography (HPLC-DAD) on a C18 column (250×4.6 mm) The mobile phasewas acetonitrile used under isocratic condition with an eluent flow rateof 1 ml/min. Standard was prepared by weighing 5 mg of standards lupeol,beta amyrin and betasitosterol (95% purity) and was made up to 10 mlwith acetonitrile and stored in darkness at 4° C. Sample was prepared byweighing 50 mg of the extract and was made up to 50 ml with acetonitrileand stored in darkness at 4° C. Both the sample and standard werefiltered separately through a 0.2 μm membrane filter before injectioninto the HPLC column. The injection volume was 20 μl. The triterpenoidswas detected at 210 nm. By comparing the area of standard and sample,the percentage of triterpenoids present in the sample was quantified.(Separation and identification of some common isomeric planttriterpenoids by thin-layer chromatography and high-performance liquidchromatography, Mitja et al, J Chromatogr A 2009 Sep. 18;1216(38):6662-70. doi: 0.1016/j.chroma.2009.07.038. Epub 2009 Jul. 29.)

Example 15 Analysis of Hydroxycinnamic Acids by HPLC

The hydroxycinnamic acids were estimated by high performance liquidchromatography (HPLC-DAD) on a C18 column (250×4.6 mm) The mobile phasewas Solvent A—1% acetic acid in water, solvent B—1% aceticacid/water/acetonitrile (2:68:30). Gradient: 0 min. 7% B increased to90% B. Flow rate 1 ml/min. Detection at 320 nm.

Standard was prepared by weighing 5 mg of standards ferulic acid andp-coumaric acid (95% purity) and was made up to 10 ml with methanol andstored in darkness at 4° C. Sample was prepared by weighing 50 mg ofextract and was made up to 50 ml with methanol and stored in darkness at4° C. Both the sample and standard were filtered separately through a0.2 μm membrane filter before injection into the HPLC column. Theinjection volume was 20 μl. The hydroxycinnamic acids were detected at320 nm. By comparing the area of standard and sample, the percentage ofhydroxycinnamic acids present in the sample was quantified. (A “NovelProtocol for the Analysis of Hydroxycinnamic Acids in Leaf Tissue ofChicory (Cichorium intybus L., Asteraceae), Meriem Bahri et al, thescientific world journal, volume 2012, Article ID 142983.)

Example 16 Analysis of Fatty Acids by GC

Fatty acids were analysed by gas chromatography method. 250 mg ofstandard oil fatty acid methyl ester was weighed in 25 ml standard flaskand made up to 25 ml with isooctane. 1 microlitre of the standardsolution was injected in GC. Retention time of each component was foundout.

0.3 gm of the sample (extract) was weighed into a 100 ml RB flask and1.5 ml of 0.5N methanolic NaOH was added. Sample was kept in a boilingwater bath with a water condenser and heated at 100° C. for 5 minute.Cooled and 2 ml of boron tri fluoride (BF3)-Methanol solution was addedand heated at 100° C. for 30 minutes. Cooled to 30-40° C. and 5 mlisooctane was added and shook vigorously for 30 seconds.

5 ml of saturated NaCl solution was added immediately and shookvigorously and cooled to room temperature. Iso-octane layer wasseparated and aqueous layer was again extracted with iso-octane.Isooctane layers were mixed and dried. Then made up the residue to 25 mlwith iso-octane and 2 micro litre was injected in GC. Retention time ofeach component was found out and compared with components of standardwith the same retention time. By comparing the area of standard andsample, the percentage of fatty acids present in the sample wasquantified. (European Pharmacopoeia, fifth edition, vol 1, ISBN:92-871-5281-0, p-110)

Example 17 Determination of Polyphenol Content

The polyphenol was estimated by high performance liquid chromatography(HPLC-DAD) on a C18 column (250×4.6 mm) The mobile phase was SolventA—0.1% Trifluro acetic acid in water, solvent B—Methanol. Isocratic(90:10) Flow rate 1 ml/min. Detection at 254 nm.

Standard was prepared by weighing 5 mg of standards gallic acid andellagic acid (95% purity) and was made up to 10 ml with methanol andstored in darkness at 4° C. Sample was prepared by weighing 50 mg of theextract and was made up to 50 ml with methanol and stored in darkness at4° C. Both the sample and standard were filtered separately through a0.2 μm membrane filter before injection into the HPLC column. Theinjection volume was 20 μl. The polyphenol was detected at 254 nm. Bycomparing the area of standard and sample, the percentage of polyphenolpresent in the sample was quantified. (HPLC Profiles of StandardPhenolic Compounds Present in Medicinal Plants, Gupta et al,International Journal of Pharmacognosy and Phytochemical Research 2012;4(3); 162-167.)

Example 18 Screening of Hypolipidemic Activity of Amla Seed ExtractUsing Triton WR 1339 Induced Dyslipidemia Model

Twenty four male Albino rats (Sprague Dawley strain) weighingapproximate 250-300 gm were selected for the study. The animals werekept in the animal house maintained at temp 24±2° C., 65% relativehumidity and 12 hr light/dark cycle. The rats were acclimatized for twoweeks and during this period they had access to standard pellet diet andwater ad libitum. After two weeks of acclimatization, all the rats werefasted overnight before injecting Triton WR 1339 (Tyloxapol) andadministration of test extracts/standard. The animals were divided intofour groups Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only)Group II: Triton WR 1339 (300 mg/kg, intraperitoneal)Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepare as per example 1 (10 mg/kg, per oral)Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byAtorvastatin (10 mg/kg, per oral)

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 1(A) Lipid profile (mg/dl) of rats treated with Amla seed extractTC TG HDL Groups Before After Before After Before After Normal 111.87134.77 52.20  62.03 37.80 58.03 control Triton only 108.3  265.6  43.06692.7  36.13 49.5  Triton + 117.2  120.87 58.63 152.23 39.4  47.27 Amlaseed extract (10 mg/Kg) Triton + 100.4  131.9  44.26 109.67 29.13 32.37Atorvastatin (10 mg/kg)

TABLE 1(B) Lipid profile (mg/dl) of rats treated with Amla seed extractLDL VLDL Groups Before After Before After Normal 63.69 64.37 10.44 12.41control Triton only 63.59 77.59 8.62 138.55 Triton + 66.03 43.15 11.7230.44 Amla seed extract (10 mg/Kg) Triton + 62.43 77.61 8.85 21.93Atorvastatin (10 mg/kg)

Results indicated that before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable. Injecting Triton alone (Group II) significantly increasedthe total cholesterol level to 265.6 mg/dl. Whereas in Group III,simultaneous oral administration of Amla seed extract in triton injectedrats, cholesterol level was 120.87 mg/dl which was 2.2 times lower ascompared to triton alone group.

In group II triton injection increased the triglyceride level to 692.7mg/dl. Whereas in Group III, simultaneous oral administration of Amlaseed extract in triton injected rats, triglyceride level was 152.23mg/dl which was 4.5 times lower as compared to triton alone group.

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.18. Whereas in Group III, simultaneous oraladministration of Amla seed extract in triton injected rats, the ratioof HDL cholesterol to total cholesterol was 0.39 which was 2.2 timeshigher as compared to triton alone group.

In group II triton injection increased the LDL level to 77.59 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract in triton injected rats, LDL level was 43.15 mg/dl which was 1.8times lower as compared to triton alone group.

In group II triton injection increased the VLDL level to 138.55 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract in triton injected rats, VLDL level was 30.44 mg/dl which was4.6 times lower as compared to triton alone group.

The standard drug Atorvastatin was also effective in lowering thecholesterol as well as triglyceride level. These results clearlyindicate the hypolipidemic activity of Amla seed extract in Tritoninduced hyperlipidemia in rats.

Example 19 Hypolipidemic Activity of Amla Seed Extracts in DifferentDoses Using Triton WR 1339 Induced Dyslipidemia Model

Sixty male Albino rats (Sprague Dawley strain) weighing approximate250-300 gm were selected for the study. The animals were kept in theanimal house maintained at temp 24±2° C., 65% relative humidity and 12hr light/dark cycle. The rats were acclimatized for two weeks and duringthis period they had access to standard pellet diet and water adlibitum. After two weeks of acclimatization, all the rats were fastedovernight before injecting Triton WR 1339 (Tyloxapol) and administrationof test extracts/standard. The animals were divided into ten groups.Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only)Group II: Triton WR 1339 (300 mg/kg, intraperitoneal)Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepare as per example 1 (10 mg/kg, per oral)Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepare as per example 1 (7.5 mg/kg, per oral)Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepare as per example 1 (5 mg/kg, per oral)Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepare as per example 1 (2.5 mg/kg, per oral)Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepare as per example 1 (2 mg/kg, per oral)Group VIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepare as per example 1 (1 mg/kg, per oral)Group IX: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepare as per example 1 (0.5 mg/kg, per oral)Group X: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byAtorvastatin (10 mg/kg, per oral)

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 2(A) Lipid profile (mg/dl) of rats treated with Amla seed extractin different doses. TC TG HDL Groups Before After Before After BeforeAfter Normal control 105.52 111.36 49.38  40.55 30.12 33.67 Triton only110.53 233.76 50.5  504.3  46.3  54.66 Triton + Amla seed 111.1  108.2351.23 91.1 45.46 48.66 extract 10 mg/Kg Triton + Amla seed 91.9 114  62.53 171.18 30.41 36.51 extract 7.5 mg/Kg Triton + Amla seed  85.46112.26 45.61 120.43 29.68 40.33 extract 5 mg/Kg Triton + Amla seed104.66 116.25 55.28 116.31 36.41 43.28 extract 2.5 mg/Kg Triton + Amlaseed  96.35 118.54 49.53 105.56 38.55 41.26 extract 2 mg/Kg Triton +Amla seed 101.25 121.55 58.33 125.56 40.25 42.58 extract 1 mg/KgTriton + Amla seed 110.66 126.61 55.48 122.28 46.26 47.41 extract 0.5mg/Kg Triton + Atorvastatin  87.43 120.83 39.9  103.86 31.66 39.83 10mg/kg

TABLE 2(B) Lipid profile (mg/dl) of rats treated with Amla seed extractin different doses. LDL VLDL Groups Before After Before After Normalcontrol 67.55 71.64 9.85 8.09 Triton only 47.46 78.24 10.09 100.86Triton + Amla seed extract 10 mg/Kg 55.4 38.76 10.24 18.13 Triton + Amlaseed extract 49.03 43.25 12.50 34.06 7.5 mg/Kg Triton + Amla seedextract 5 mg/Kg 46.68 47.85 9.12 24.08 Triton + Amla seed extract 57.2349.73 11.05 23.26 2.5 mg/Kg Triton + Amla seed extract 2 mg/Kg 47.956.17 9.90 21.11 Triton + Amla seed extract 1 mg/Kg 49.34 53.86 11.6625.11 Triton + Amla seed extract 53.31 54.75 11.09 24.45 0.5 mg/KgTriton + Atorvastatin 10 mg/kg 47.75 60.23 7.98 20.77

Results indicated that intraperitoneal injection of Triton significantlyincreased the total cholesterol level about 2 times over the baselinevalue. Similarly, triglyceride level also increased to very high level(Group II) following Triton injection. Simultaneous oral administrationof various doses of Amla seed extract in triton injected rats loweredthe cholesterol increase to almost normal level (Group III to IX).Triglyceride level was also lowered by all the doses of Triton with amlaseed extract as compared to Triton alone group. The standard drugAtorvastatin was also effective in lowering the cholesterol as well astriglyceride level (Group X). These results clearly indicate thehypolipidemic activity of Amla seed extract at various dose levels inTriton induced hyperlipidemia in rats.

Example 20 Hypolipidemic Activity of Amla Seed Extract Compared withOther Amla Extracts Using Triton WR 1339 Induced Dyslipidemia Model

Forty eight male Albino rats (Sprague Dawley strain) weighingapproximate 250-300 gm were selected for the study. The animals werekept in the animal house maintained at temp 24±2° C., 65% relativehumidity and 12 hr light/dark cycle. The rats were acclimatized for twoweeks and during this period they had access to standard pellet diet andwater ad libitum. After two weeks of acclimatization, all the rats werefasted overnight before injecting Triton WR 1339 (Tyloxapol) andadministration of test extracts/standard. The animals were divided intoeight groups. Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only)Group II: Triton WR 1339 (300 mg/kg, intraperitoneal) Group III: TritonWR 1339 (300 mg/kg, intraperitoneal) followed by Amla seed extractprepared as per example 1 (2.5 mg/kg, per oral)Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaextract prepared as per example 3 (40 mg/kg, per oral)Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaextract prepared as per example 4 (40 mg/kg, per oral)Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaextract prepared as per example 5 (40 mg/kg, per oral)Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaextract prepared as per example 6 (40 mg/kg, per oral)Group VIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byAtorvastatin (10 mg/kg, per oral)

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 3(A) Lipid profile (mg/dl) of rats treated with Amla seed extractand Amla fruit extracts TC TG HDL Groups Before After Before AfterBefore After Normal control  95.52  99.36 42.38  48.51 31.02 32.11Triton only 102.52 348.87 61.51 614.21 41.90 51.22 Triton + Amla seed108.22 112.23 61.23  95.88 39.46 47.51 extract prepared as per example 1(2.5 mg/Kg) Triton + Amla  96.52 128.25 54.55 102.34 44.25 48.35 extractprepared as per example 3 (40 mg/kg) Triton + Amla 108.24 124.55 59.32115.36 41.26 46.52 extract prepared as per example 4 (40 mg/kg) Triton +Amla 100.66 135.36 65.25 132.22 38.66 40.11 extract prepared as perexample 5 (40 mg/kg) Triton + Amla  97.56 132.66 60.21 125.54 34.5936.54 extract prepared as per example 6 (40 mg/kg) Triton +  97.33132.58 48.99 218.66 35.11 33.84 Atorvastatin (10 mg/kg)

TABLE 3(B) Lipid profile (mg/dl) of rats treated with Amla seed extractand Amla fruit extracts LDL VLDL Groups Before After Before After Normalcontrol 56.03 57.54 8.47 9.70 Triton only 48.32 174.80 12.30 122.84Triton + Amla seed 56.52 45.55 12.24 19.17 extract prepared as perexample 1 (2.5 mg/Kg) Triton + Amla extract 41.36 59.43 10.91 20.46prepared as per example 3 (40 mg/kg) Triton + Amla extract 55.11 54.9511.86 23.07 prepared as per example 4 (40 mg/kg) Triton + Amla extract48.95 68.80 13.05 26.44 prepared as per example 5 (40 mg/kg) Triton +Amla extract 50.92 71.01 12.04 25.10 prepared as per example 6 (40mg/kg) Triton + Atorvastatin 52.43 55.00 9.79 43.73 (10 mg/kg)

In this study, effectiveness of Amla seed extract (2.5 mg of extract/kgof subject) was compared with other amla extracts made as in Examples 3to 6.

Results indicated that before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable. Injecting Triton alone (Group II) significantly increasedthe total cholesterol level to 348.87 mg/dl. Whereas in Group III,simultaneous oral administration of Amla seed extract in triton injectedrats, cholesterol level was 112.23 mg/dl which was 3.1 times lower ascompared to triton alone group.

In group II triton injection increased the triglyceride level to 614.21mg/dl. Whereas in Group III, simultaneous oral administration of Amlaseed extract in triton injected rats, triglyceride level was 95.88 mg/dlwhich was 6.4 times lower as compared to triton alone group.

In triton alone group after triton injection, the ratio of HDL to totalcholesterol was 0.14. Whereas in Group III, simultaneous oraladministration of Amla seed extract in triton injected rats, the ratioof HDL cholesterol to total cholesterol was 0.42 which was 3 timeshigher as compared to triton alone group.

In group II triton injection increased the LDL level to 174.8 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract in triton injected rats, LDL level was 45.55 mg/dl which was 3.8times lower as compared to triton alone group.

In group II triton injection increased the VLDL level to 122.84 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract in triton injected rats, VLDL level was 19.17 mg/dl which was6.4times lower as compared to triton alone group.

The other amla extracts (prepared in example 3 to 6) at 40 mg/kg werealso able to decrease the cholesterol and triglyceride level but thesewere less effective than amla seed extract prepared in example 1. Thestandard drug Atorvastatin was also effective in lowering thecholesterol as well as triglyceride levels (Group VIII).

Example 21 Single Dosage Study for Evaluating Hypolipidemic Activity ofAmla Seed Extract Compared with Other Amla Extracts Using Triton WR 1339Induced Dyslipidemia Model

Forty eight male Albino rats (Sprague Dawley strain) weighingapproximate 250-300 gm were selected for the study. The animals werekept in the animal house maintained at temp 24±2° C., 65% relativehumidity and 12 hr light/dark cycle. The rats were acclimatized for twoweeks and during this period they had access to standard pellet diet andwater ad libitum. After two weeks of acclimatization, all the rats werefasted overnight before injecting Triton WR 1339 (Tyloxapol) andadministration of test extracts/standard. The animals were divided intoeight groups. Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only)Group II: Triton WR 1339 (300 mg/kg, intraperitoneal)Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaseed extract prepared as per example 1 (2.5 mg/kg, per oral)Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaextract prepared as per example 3 (2.5 mg/kg, per oral)Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaextract prepared as per example 4 (2.5 mg/kg, per oral)Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaextract prepared as per example 5 (2.5 mg/kg, per oral)Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Amlaextract prepared as per example 6 (2.5 mg/kg, per oral)Group VIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byAtorvastatin (10 mg/kg, per oral)

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 4(A) Lipid profile (mg/dl) of rats treated with Amla seed extractand Amla fruit extracts in same dosage level TC TG HDL Groups BeforeAfter Before After Before After Normal control 102.25 101.56 56.33 54.9639.65 42.51 Triton only 91.55 385.54 55.69 644.65 44.52 59.54 Triton +94.66 108.25 60.23 111.36 38.41 46.23 Amla seed extract prepared as perexample 1 (2.5 mg/Kg) Triton + 100.58 145.24 51.63 140.63 41.55 42.65Amla extract prepared as per example 3 (2.5 mg/kg) Triton + 106.52139.66 49.36 131.33 42.36 46.31 Amla extract prepared as per example 4(2.5 mg/kg) Triton + 95.66 159.65 68.55 151.33 35.65 37.63 Amla extractprepared as per example 5 (2.5 mg/kg) Triton + 98.69 152.36 67.96 148.6537.55 39.54 Amla extract prepared as per example 6 (2.5 mg/kg) Triton +99.83 122.54 46.59 211.06 39.51 36.66 Atorvastatin (10 mg/kg)

TABLE 4(B) Lipid profile (mg/dl) of rats treated with Amla seed extractand Amla fruit extracts in same dosage level LDL VLDL Groups BeforeAfter Before After Normal control 51.33 48.06 11.26 10.99 Triton only35.89 197.07 11.13 128.93 Triton + Amla seed 44.20 39.75 12.04 22.27extract prepared as per example 1 (2.5 mg/Kg) Triton + Amla extract48.70 74.47 10.32 28.12 prepared as per example 3 (2.5 mg/kg) Triton +Amla extract 54.28 67.09 9.87 26.26 prepared as per example 4 (2.5mg/kg) Triton + Amla extract 46.3 91.76 13.71 30.26 prepared as perexample 5 (2.5 mg/kg) Triton + Amla extract 47.54 83.09 13.59 29.73prepared as per example 6 (2.5 mg/kg) Triton + 51.00 43.67 9.31 42.21Atorvastatin(10 mg/kg

In this study, Amla seed extract (2.5 mg extract/kg of subject) wascompared with other amla extracts made as in example 3 to 6 at the samedose level, i.e. 2.5 mg of extract/kg of subject.

Results indicated that before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable. Injecting Triton alone (Group II) significantly increasedthe total cholesterol level to 385.54 mg/dl. Whereas in Group III,simultaneous oral administration of Amla seed extract in triton injectedrats, cholesterol level was 108.25 mg/dl which was 3.6 times lower ascompared to triton alone group.

In group II triton injection increased the triglyceride level to 644.65mg/dl. Whereas in Group III, simultaneous oral administration of Amlaseed extract in triton injected rats, triglyceride level was 111.36mg/dl which was 5.8 times lower as compared to triton alone group.

In triton alone group after triton injection, the ratio of HDL to totalcholesterol was 0.15. Whereas in Group III, simultaneous oraladministration of Amla seed extract in triton injected rats, the ratioof HDL cholesterol to total cholesterol was 0.42 which was 2.8 timeshigher as compared to triton alone group.

In group II triton injection increased the LDL level to 197.07 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract in triton injected rats, LDL level was 39.75 mg/dl which was 4.9times lower as compared to triton alone group.

In group II triton injection increased the VLDL level to 128.93 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract in triton injected rats, VLDL level was 22.27 mg/dl which was5.8 times lower as compared to triton alone group

The other amla extracts (prepared in example 3 to 6) at 2.5 mg/kg wereable to decrease the cholesterol and triglyceride level to a certainextent only but these are not as effective as amla seed extract at samedose levels. The studies carried out indicate that the standard drugAtorvastatin though was effective in lowering the cholesterol as well astriglyceride levels (Group VIII) but less effective when compared withthe amla seed extract.

Example 22 Antidiabetic Activity of Amla Seed Extract in StreptozotocinInduced Diabetic Rats

The Amla seed extract as prepared in example 1 was evaluated forantidiabetic activity in experimental rats. Male/Female albino wistarrats were maintained as per standard guidelines: housed in polypropylenecages, under 12 hour artificial light and dark cycles at a temperatureof 24±2° C., given a standard pellet diet and water ad libitum. Theanimals were acclimatized to the animal house conditions for a weekbefore beginning the experiment.

Diabetes was induced by injecting streptozotocin 35 mg/kg dissolved in0.1M citrate buffer of pH 4.5, intra-peritoneally. Five days afterinduction of diabetes (day 1 of the study), animals were fasted for 12hours and the fasting blood glucose level (FBG) was estimated fordiagnosing diabetic rats. Animals with FBG above 200 mg/dl wereconsidered diabetic. The diabetic animals were randomly divided intothree groups of six animals each.

Following table 5 shows the treatment schedule given to the respectivegroup of animals for 28 days.

TABLE 5 Treatment schedule Groups Drugs administered Group I Vehicle for28 days Group II Glibenclamide (0.5 mg/kg) for 28 days Group III Amlaseed extract as per Example 1 (10 mg/kg) for 28 days

Fasting blood glucose level and body weight of rats was measuredinitially and then at Day 7, Day 14, Day 21 and Day 28 of the study. Theplasma CRP level was measured initially and then at Day 28 of the study.

TABLE 6 Fasting blood glucose (FBG) level of diabetic rats treated withamla seed extract Fasting Blood Glucose level (mg/dl) Day 1 Day Day DayDay Groups Treatment (Initial) 7 14 21 28 Group I Vehicle 355 382 391411 431 Group II Glibenclamide 406 211 179 136 109 Group III Amla seedextract 369 266 211 172 135 as per Example 1

TABLE 7 CRP level of diabetic rats treated with amla seed extract CRPlevel (mg/L) Day 1 Day 28 Groups Treatment (Initial) (Final) Group IVehicle 10.3 11.3 Group II Glibenclamide 10.8 9.6 Group III Amla seedextract as per Example 1 9.9 6.3

As shown in the results, a single ip injection of streptozotocinincreased the blood glucose level to very high level and made the ratsdiabetic. Treatment of diabetic rats with amla seed extractsignificantly lowered the blood glucose level in 28 days. Thatcorresponds to a 2.7 times reduction in fasting blood glucose level indiabetic rats as compared to baseline (Day 1) after administration ofamla seed extract for 28 days. Treatment with glibenclamide alsodecreased the blood glucose level to nearly normal in 28 days. Bodyweight of diabetic rats treated with amla seed extract recoveredsignificantly as compared to vehicle group. The CRP level decreasedsignificantly in amla seed extract fed group but glibenclamide failed todecrease the CRP level significantly as compared to initial CRP value.28 days treatment with Amla seed extract to diabetic rats decreased theCRP level 1.6 times than initial level.

Example 23 Hypolipidemic Activity of Amla Seed Extract in CholesterolFed Rabbits

The Amla seed extract as prepared in example 1 was evaluated forhypolipidemic activity in experimental rats. Male NZ white rabbitsweighing 1.5-2.0 kg were used for the experiment. They were housed in atemperature-controlled room (25±2° C.) in clean stainless steel cageswith ‘12 h light and 12 h dark’ cycles and fed with normal pellet dietand water ad libitum.

After the acclimatization period of 10 days, blood samples werecollected from marginal ear vein of all the rabbits. The blood wasallowed to clot and then centrifuged at 3000 rpm for 10 min and theserum was carefully drawn and collected into separate tubes. The serumwas analyzed for total cholesterol, triglycerides, high densitylipoproteins (HDL), low density lipoproteins (LDL) and very low densitylipoproteins (VLDL) levels using auto-analyzer.

After taking baseline lipid profile the animals were divided into fourgroups comprising of six animals in each group. Following treatment wasgiven to the animals for the three months:

TABLE 8 Treatment schedule Groups Drugs administered Group I Vehicle for3 months Group II Cholesterol (100 mg/kg) + Vehicle for 3 months GroupIII Cholesterol (100 mg/kg) + Amla seed extract as per example 1 (10mg/kg) for 3 months Group IV Cholesterol (100 mg/kg) + Atorvastatin (10mg/kg) for 3 months

Blood samples were collected from all the rabbits after 3 months oftreatment and serum was analyzed for lipid profile. After bloodcollection the animals were sacrificed after injecting pentobarbitoneand aorta was dissected out, washed with saline and preserved in 10%formalin for histopathology. The sections were cut using microtome andstained with heamatoxylin & eosin dye and mounted on glass slides. Theslides were observed under microscope and aortic intima media thickness(IMT) was measured by histomorphometry.

TABLE 9 (A) Lipid profile (mg/dl) of rabbits treated with amla seedextract TC TG HDL Groups Before After Before After Before After Normalcontrol 58.23 56.77 62.25 60.05 16.81 15.89 (vehicle only) Cholesterol +54.88 219.81 59.26 188.25 14.58 11.5 Vehicle Cholesterol + 56.36 94.1860.61 83.23 15.42 37.27 Amla seed extract Cholesterol + 53.69 118.958.28 78.67 14.15 20.37 Atorvastatin

TABLE 9 (B) Lipid profile (mg/dl) of rabbits treated with amla seedextract LDL VLDL Groups Before After Before After Normal control 28.9728.37 12.45 12.01 (vehicle only) Cholesterol + 28.45 170.66 11.85 37.65Vehicle Cholesterol + Amla 28.82 40.26 12.12 16.65 seed extractCholesterol + 27.88 82.8 11.66 15.73 Atorvastatin

TABLE 10 Aortic intima media thickness (IMT) of rabbits treated withamla seed extract Aortic intima media thickness Groups (μm) Normalcontrol (vehicle only) 49.52 Cholesterol + Vehicle 123.24 Cholesterol +Amla seed extract 66.21 Cholesterol + Atorvastatin 112.36

Results show that, before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable. Feeding cholesterol for three months increased the totalcholesterol and triglyceride levels to very high.

Administration of Cholesterol alone (Group II) significantly increasedthe total cholesterol level to 219.81 mg/dl. Whereas in Group III, oraladministration of cholesterol+Amla seed extract, total cholesterol levelwas 94.18 mg/dl which was 2.3 times lower as compared to cholesterolalone group.

In group II cholesterol administration increased the triglyceride levelto 188.25 mg/dl. Whereas in Group III, oral administration ofcholesterol+Amla seed extract, triglyceride level was 83.23 mg/dl whichwas 2.3 times lower as compared to cholesterol alone group.

In group III, administration of cholesterol+Amla seed extract increasedthe HDL (good cholesterol) level significantly from a baseline value of15.42 to 37.27 after 3 months treatment. That was a 2.4 times increasein the HDL cholesterol after 3 month treatment.

After cholesterol administration the cholesterol alone group showed aratio of HDL to total cholesterol was 0.05. Whereas in Group III, oraladministration of cholesterol+Amla seed extract, the ratio of HDLcholesterol to total cholesterol was 0.39 which was 7.8 times higher ascompared to cholesterol alone group.

In group II administration of cholesterol alone increased the LDL levelto 170.66 mg/dl. Whereas in Group III, oral administration ofcholesterol+Amla seed extract, LDL level was 40.26 mg/dl which was 4.2times lower as compared to triton alone group.

In group II administration of cholesterol alone increased the VLDL levelto 37.65 mg/dl. Whereas in Group III, oral administration ofcholesterol+Amla seed extract, VLDL level was 16.65 mg/dl which was 2.3times lower as compared to cholesterol alone group

Atorvastatin also decreased the total cholesterol and triglyceride levelsignificantly as compared to untreated control group but amla seedextract was better especially in increasing the HDL level.

Moreover, administration of amla seed extract showed a 1.9 timesreduction in the intima media thickness of aorta of rabbits as comparedto untreated control group.

Example 24 Hair Fall Prevention and Hair Growth Promoting Activity ofAmla Seed Extract in Humans Method:

10 human subjects suffering with alopecia and severe hair fall (asdetected by a dermatologist) were randomly divided into two groupscomprising of 5 subjects in each.

Group I—Amla seed extract group prepared as per example 1Group II—Placebo group

All the subjects were prohibited to take any kind of medicines (oral ortopical) having hair growth promotion like minoxidil, finasteride etc.for one month prior to the study initiation. The subjects of group Iapplied 5 ml coconut oil containing 5% amla seed extract twice daily onthe affected area and also took 100 mg of amla seed extract capsulestwice daily. The subjects of placebo group were given coconut oil(without amla seed extract) to be applied twice daily on affected areaand were provided with placebo capsules to be taken twice daily. Thetreatment was continued for 3 months and observations were taken by adermatologist before and after the study period. Length and thickness ofrandomly plucked five hairs from each subject was also determined.

Results:

The hairs of subjects treated with amla seed extract were shiny,lustrous and denser as compared to placebo group. The hair fall wasalmost arrested in the subjects treated with amla seed extract. Incontrast, the subjects of placebo group observed hair fall at the samerate as it was before starting the treatment. The average length ofhairs of subjects treated with amla seed extract was about 25% more thanthe subjects of placebo group. Thickness of the hairs was also about 20%more in amla seed extract group as compared to subjects in placebogroup. Therefore, amla seed extract was helpful in decreasing the hairfall as well as in promoting hair growth. Amla seed extract also madethe hairs lustrous and shiny.

Example 25 Hypolipidemic Activity of Amla Seed Extract Compared withOther Amla Extracts Using Triton WR 1339 Induced Dyslipidemia Model

Forty eight male Albino rats (Sprague Dawley strain) weighingapproximate 250-300 gm were selected for the study. The animals werekept in the animal house maintained at temp 24±2° C., 65% relativehumidity and 12 hr light/dark cycle. The rats were acclimatized for twoweeks and during this period they had access to standard pellet diet andwater ad libitum. After two weeks of acclimatization, all the rats werefasted overnight before injecting Triton WR 1339 (Tyloxapol) andadministration of test extracts/standard. The animals were divided intotwelve groups. Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only)Group II: Triton WR 1339 (300 mg/kg, intraperitoneal)Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Ethylacetate of methanol extract of Amla seed prepared as per example 1 (2.5mg/kg, per oral)Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by waterpart of methanol extract of Amla seed prepared as per example 1 (2.5mg/kg, per oral)Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by ethylacetate extract of Amla seed prepared as per example 2 (2.5 mg/kg, peroral)Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by powderof dried Amla seed prepared as per example 7 (2.5 mg/kg, per oral)Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by waterextract of dried Amla seed powder prepared as per example 8 (2.5 mg/kg,per oral)Group VIII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed bymethanol extract of dried Amla seed powder prepared as per example 8(2.5 mg/kg, per oral)Group IX: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by powderof dried amla fruit prepared as per example 10 (2.5 mg/kg, per oral)Group X: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by waterextract of dried Amla fruit powder prepared as per example 11 (2.5mg/kg, per oral)Group XI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed bymethanol extract of dried Amla fruit powder prepared as per example 12(2.5 mg/kg, per oral)Group XII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byAtorvastatin (2.5 mg/kg, per oral)

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hour of drug treatment 2 ml blood sampleswere collected from the retro orbital plexus. The blood was allowed toclot and then centrifuged at 3000 rpm for 10 min and the serum wascarefully drawn and collected into separate tubes. The serum wasanalyzed for total cholesterol, triglycerides, high density lipoproteins(HDL), low density lipoproteins (LDL) and very low density lipoproteins(VLDL) levels using auto-analyzer.

TABLE 11(A) Lipid profile (mg/dl) of rats treated with Amla seed extractand Amla fruit extracts in same dosage level TC TG HDL Groups BeforeAfter Before After Before After Normal control 90.15 94.16 58.18 61.6530.12 31.01 Triton only 98.92 288.18 60.15 581.42 35.59 42.62 Triton +EtOAc 101.12 106.41 62.42 90.47 34.14 48.15 extract Amla seed preparedas per example 1 (2.5 mg/Kg) Triton + Water 102.54 266.25 54.57 556.3532.24 37.54 part of Amla seed prepared as per example 1 (2.5 mg/kg)Triton + EtOAc 99.35 122.25 55.87 99.5 36.25 42.51 extract of Amla seedprepared as per example 2 (2.5 mg/kg) Triton + powder 97.54 275.56 58.65541.35 31.25 36.65 of dried Amla seed prepared as per example 7 (2.5mg/kg) Triton + water 104.55 284.66 61.23 571.44 33.14 38.51 extract ofAmla seed powder prepared as per example 8 (2.5 mg/kg) Triton + methanol96.55 251.64 55.69 552.36 35.68 39.41 extract of Amla seed powderprepared as per example 9 (2.5 mg/kg) Triton + powder of 95.64 253.1459.68 575.36 33.54 37.74 dried Amla fruit prepared as per example 10(2.5 mg/kg) Triton + water 101.22 265.33 59.85 547.96 31.54 41.52extract of Amla fruit powder prepared as per example 11 (2.5 mg/kg)Triton + methanol 99.64 254.39 60.77 577.63 34.88 40.57 extract of Amlaseed powder prepared as per example 12 (2.5 mg/kg) Triton + 104.44123.14 49.54 195.25 36.54 39.56 Atorvastatin (2.5 mg/kg)

TABLE 11(B) Lipid profile (mg/dl) of rats treated with Amla seed extractand Amla fruit extracts in same dosage level LDL VLDL Groups BeforeAfter Before After Normal control 48.39 50.82 11.64 12.33 Triton only51.3 129.28 12.03 116.28 Triton + EtOAc extract Amla seed 54.5 40.1712.48 18.09 prepared as per example 1 (2.5 mg/Kg) Triton + Water part ofAmla seed 59.39 117.44 10.91 111.27 prepared as per example 1 (2.5mg/kg) Triton + EtOAc extract of Amla 51.93 59.84 11.17 19.9 seedprepared as per example 2 (2.5 mg/kg) Triton + powder of dried Amla54.56 130.64 11.73 108.27 seed prepared as per example 7 (2.5 mg/kg)Triton + water extract of Amla 59.16 131.86 12.25 114.29 seed powderprepared as per example 8 (2.5 mg/kg) Triton + methanol extract of Amla49.73 101.76 11.14 110.47 seed powder prepared as per example 9 (2.5mg/kg) Triton + powder of dried Amla 50.16 100.33 11.94 115.07 fruitprepared as per example 10 (2.5 mg/kg) Triton + water extract of Amla57.71 114.22 11.97 109.59 fruit powder prepared as per example 11 (2.5mg/kg) Triton + methanol extract of Amla 52.61 98.29 12.15 115.53 seedpowder prepared as per example 12 (2.5 mg/kg) Triton + Atorvastatin (2.5mg/kg) 57.99 44.53 9.91 39.05

In this study, Amla seed extract (2.5 mg extract/kg body weight) made inexample 1 and 2 were compared with other amla extracts made as inexample 7 to 12 at the same dose level, i.e. 2.5 mg of extract/kg bodyweight.

Results show that, before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable.

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 288.18 mg/dl. Whereas in Group III, simultaneousoral administration of Amla seed extract prepared as per example 1 intriton injected rats, cholesterol level was 106.41 mg/dl which was 2.7times lower as compared to triton alone group.

In group II triton injection increased the triglyceride level to 581.42mg/dl. Whereas in Group III, simultaneous oral administration of Amlaseed extract prepared as per Example 1 in triton injected rats,triglyceride level was 90.47 mg/dl which was 6.4 times lower as comparedto triton alone group.

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.15. Whereas in Group III, simultaneous oraladministration of Amla seed extract prepared as per Example 1 in tritoninjected rats, the ratio of HDL cholesterol to total cholesterol was0.45 which was 3 times higher as compared to triton alone group.

In group II triton injection increased the LDL level to 129.28 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract (prepares as per example 1) in triton injected rats, LDL levelwas 40.17 mg/dl which was 3.2 times lower as compared to triton alonegroup.

In group II triton injection increased the VLDL level to 116.28 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract (prepared as per example 1) in triton injected rats, VLDL levelwas 18.09 mg/dl which was 6.4 times lower as compared to triton alonegroup

The ethyl acetate extract of amla seeds (prepared in example 2) at 2.5mg/kg were also able to decrease the cholesterol and triglyceride levelto a certain extent (Group V).

Results show that, before treatment the baseline value of totalcholesterol, triglyceride, HDL, LDL and VLDL in all groups werecomparable.

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 288.18 mg/dl. Whereas in Group III, simultaneousoral administration of Amla seed extract prepared as per example 2 intriton injected rats, cholesterol level was 122.25 mg/dl which was 2.3times lower as compared to triton alone group.

In group II triton injection increased the triglyceride level to 581.42mg/dl. Whereas in Group III, simultaneous oral administration of Amlaseed extract prepared as per Example 2 in triton injected rats,triglyceride level was 99.5 mg/dl which was 5.8 times lower as comparedto triton alone group.

In triton alone group after injection the ratio of HDL to totalcholesterol was 0.15. Whereas in Group III, simultaneous oraladministration of Amla seed extract prepared as per example 2 in tritoninjected rats, the ratio of HDL cholesterol to total cholesterol was0.35 which was 2.3 times higher as compared to triton alone group.

In group II triton injection increased the LDL level to 129.28 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract (prepares as per example 2) in triton injected rats, LDL levelwas 59.84 mg/dl which was 2.1 times lower as compared to triton alonegroup.

In group II triton injection increased the VLDL level to 116.28 mg/dl.Whereas in Group III, simultaneous oral administration of Amla seedextract (prepared as per example 2) in triton injected rats, VLDL levelwas 19.9 mg/dl which was 5.8 times lower as compared to triton alonegroup

The water part of methanol extract (prepared in example 1), amla seedpowder and other water and methanol extracts (prepared in example 7 to12) were not active in reducing cholesterol or triglyceride levels.Though the standard drug Atorvastatin was effective in lowering thecholesterol as well as triglyceride levels (Group XII) but lesseffective when compared with the ethyl acetate part of amla seedmethanol extract (as per example 1) or ethyl acetate extract of amlaseeds (as per example 2).

Example 26 Hypolipidemic Activity of Amla Seed Extract (Product 3)Compared with Amla Seed Powder Using Triton WR 1339 Induced DyslipidemiaModel

Twenty eight male Albino rats (Sprague Dawley strain) weighingapproximately 250-300 gm were selected for the study. The animals weremaintained at temperature of 24±2° C., 65% relative humidity and 12 hrlight/dark cycle. The rats were acclimatized for two weeks and duringthis period they had access to standard pellet diet and water adlibitum. After two weeks of acclimatization, all the rats were fastedovernight before injecting Triton WR 1339 (Tyloxapol) and administrationof test extracts/standard. The animals were divided into seven groups.Following treatment was given to overnight fasted rats:

Group I: Normal control (vehicle only)Group II: Triton WR 1339 (300 mg/kg, intraperitoneal)Group III: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byadministering product of Amla seed extract (Product 3) on the same dayof triton administration (0.5 mg/kg, per oral)Group IV: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byProduct 2 (50% methanol fraction+80% methanol fraction+water fraction)of Amla seed extract on the same day that Triton was adminstered (0.5mg/kg, per oral)Group V: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by Product1 (Hexane fraction) of Amla seed extract on the same day that Triton wasadministered (0.5 mg/kg, per oral)Group VI: Triton WR 1339 (300 mg/kg, intraperitoneal) followed by powderof dried Amlaseed prepared as per example 8 (0.5 mg/kg, per oral). Thepowder of dried Amla seed was administered on the same day as Triton wasadministered.Group VII: Triton WR 1339 (300 mg/kg, intraperitoneal) followed byAtorvastatin administration (10 mg/kg, per oral), on the same day.

The animals were deprived of food for next 24 hours but had free accessto water ad libitum. After 24 hours of drug treatment, 2 ml of bloodsample was collected from the retro orbital plexus. The blood wasallowed to clot and then centrifuged at 3000 rpm for 10 min and theserum was carefully drawn and collected into separate tubes. The serumwas analyzed for total cholesterol, triglycerides, high densitylipoproteins (HDL), low density lipoproteins (LDL) and very low densitylipoproteins (VLDL) levels using auto-analyzer.

TABLE 12(A) Lipid profile (mg/dl) of rats treated with Amla seedextracts (product3) and different fractions (Product1 and product 2) TCTG HDL Groups Before After Before After Before After Group I 91.05 95.1157.16 60.47 29.31 30.11 Normal control Group II 109.55 386.4 49.5 583.3332.5 43.2 Triton only Group III 90.2 95.6 44.66 62.4 28.78 49.36Triton + Amla seed Product 3 (0.5 mg/Kg) Group IV 96.5 132.36 46.8 103.527.4 41.03 Triton + Amla seed Product 2 (0.5 mg/Kg) Group V 95.7 184.550.12 119.4 30.9 47.97 Triton + Amla seed Product 1 (0.5 mg/Kg) Group VI96.85 345.6 59.65 542.54 32.35 34.45 Triton + powder of dried Amla seedprepared as per example 8 (0.5 mg/kg) Group VII 103.47 121.21 48.54192.25 35.54 38.46 Triton + Atorvastatin (10 mg/kg)

TABLE 12(B) Lipid profile (mg/dl) of rats treated with Amla seedextracts (product 3) and different fractions (Product1 and product 2)LDL VLDL Groups Before After Before After Group I 50.31 52.91 50.3152.91 Normal control Group II 67.15 226.53 67.15 226.53 Triton onlyGroup III 60.81 33.76 60.81 33.76 Triton + Amla seed Product 3 (0.5mg/Kg) Group IV 62.3 70.63 62.3 70.63 Triton + Amla seed Product 2 (0.5mg/Kg) Group V 65.4 112.65 65.4 112.65 Triton + Amla seed Product 1 (0.5mg/Kg) Group VI 52.57 202.64 52.57 202.64 Triton + powder of dried Amlaseed prepared as per example 8 (0.5 mg/kg) Group VII 58.22 44.30 58.2244.30 Triton + Atorvastatin (10 mg/kg)

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 386.4 mg/dl. Whereas in Group III, following oraladministration of Product 3 of Amla seed extract to triton injectedrats, the cholesterol level was 95.6 mg/dl. Therefore, administeringProduct 3 (Group III) resulted in 4 times lower level of cholesterol ascompared to triton alone treatment (Group II).

In Group II triton injection increased the triglyceride level to 583.33mg/dl. Whereas in Group III, oral administration of Product 3 to tritoninjected rats, the triglyceride level was 62.4 mg/dl, which was 9.3times lower as compared to triton alone group (Group 2).

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.11. Whereas in Group III, the ratio of HDL cholesterolto total cholesterol was 0.51, which was 4.6 times higher as compared totriton alone group.

In group II triton injection increased the LDL level to 226.53 mg/dl.Whereas in Group III, LDL level was 33.76 mg/dl, which was 6.7 timeslower as compared to triton alone group.

In group II triton injection increased the VLDL level to 116.67 mg/dl.Whereas in Group III, the VLDL level was 12.48 mg/dl, which was 9.3times lower as compared to triton alone group.

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 386.4 mg/dl. Whereas in Group IV (Triton followedby Product 2 administration), cholesterol level was 132.36 mg/dl, whichwas 2.9 times lower as compared to triton alone group.

In Group II Triton injection increased the triglyceride level to 583.33mg/dl. Whereas in Group IV, oral administration of Product 2 to tritoninjected rats, triglyceride level was 103.5 mg/dl, which was 5.6 timeslower as compared to triton alone group.

In triton alone group after triton injection the ratio of HDL to totalcholesterol was 0.11. Whereas in Group IV, oral administration of Amlaseed extract (Product 2) to triton injected rats, the ratio of HDLcholesterol to total cholesterol was 0.30 which was 2.7 times higher ascompared to triton alone group.

In Group II, Triton injection increased the LDL level to 226.53 mg/dl.Whereas in Group IV, oral administration of Product 2 to triton injectedrats, LDL level was 70.63 mg/dl, which was 3.2 times lower as comparedto triton alone group.

In Group II, triton injection increased the VLDL level to 116.67 mg/dl.Whereas in Group IV, oral administration of Product 2 to triton injectedrats, VLDL level was 20.7 mg/dl, which was 5.6 times lower as comparedto triton alone group (Group II).

Injecting Triton alone (Group II) significantly increased the totalcholesterol level to 386.4 mg/dl. Whereas in Group V, simultaneous oraladministration of product 1 prepared as per example 13 in tritoninjected rats, cholesterol level was 184.5 mg/dl which was 2.09 timeslower as compared to triton alone group.

In group II triton injection increased the triglyceride level to 583.33mg/dl. Whereas in Group V, simultaneous oral administration of product 1in triton injected rats, triglyceride level was 119.4 mg/dl which was4.9 times lower as compared to triton alone group.

In triton alone group after injection the ratio of HDL to totalcholesterol was 0.11. Whereas in Group V, simultaneous oraladministration of product 1 prepared as per example 13 in tritoninjected rats, the ratio of HDL cholesterol to total cholesterol was0.26 which was 2.4 times higher as compared to triton alone group.

In group II triton injection increased the LDL level to 226.53 mg/dl.Whereas in Group V, simultaneous oral administration of product 1prepared as per example 13 in triton injected rats, LDL level was 112.65mg/dl which was 2 times lower as compared to triton alone group.

In group II triton injection increased the VLDL level to 116.67 mg/dl.Whereas in Group V, simultaneous oral administration of product 1prepared as per example 13 in triton injected rats, VLDL level was 23.88mg/dl which was 4.9 times lower as compared to triton alone group.

Other modifications and variations to the invention will be apparent tothose skilled in the art from the foregoing disclosure and teachings.Thus, while only certain embodiments of the invention have beenspecifically described herein, it will be apparent that numerousmodifications may be made thereto without departing from the spirit andscope of the invention.

What is claimed is:
 1. An amla seed blend comprising a blend of Product1 and Product 2, wherein the Product 1 comprises alpha linolenic acid,linoleic acid and oleic acid, wherein the Product 2 comprisestriterpenoids and hydroxycinnamic acids, wherein a ratio of Product 2 toProduct 1 ranges from about 1:60 to about 99:1.
 2. The amla seed blendof claim 1 comprising about 6% to about 50% of triterpenoids, about 2%to about 20% of hydroxycinnamic acids, about 10% to about 60% of fattyacids, wherein the triterpenoids comprises beta-sitosterol, beta amyrinand lupeol, wherein the hydroxycinnamic acids comprises ferulic acid andp-coumaric acid, and wherein the fatty acid comprises alpha linolenicacid, linoleic acid and oleic acid.
 3. A dosage form comprising the amlaseed blend of claim 1, the dosage form comprising capsule, tablet,granule, sachet, powder, paste, ointment, infusion, injection, ampoule,solution, suspension, emulsion, pills, oil, and cream.
 4. The amla seedblend of claim 1, wherein the ratio of Product 2 to Product 1 rangesfrom about 1:1 to about 99:1.
 5. The amla seed blend of claim 1, whereinthe ratio of Product 2 to Product 1 ranges from about 1:1 to about 1:10.6. The amla seed blend of claim 1, wherein the ratio of Product 2 toProduct 1 ranges from about 1:10 to 10:1.
 7. The amla seed blend ofclaim 1, wherein the ratio of Product 2 to Product 1 is about 2:3. 8.The amla seed blend of claim 1, wherein the ratio of Product 2 toProduct 1 is about 1:2.
 9. The amla seed blend of claim 1, wherein theratio of Product 2 to Product 1 is about 1:1.
 10. The amla seed blend ofclaim 1, wherein the ratio of Product 2 to Product 1 is about 3:2. 11.The amla seed blend of claim 1, wherein the ratio of Product 2 toProduct 1 is about 10:1.
 12. The amla seed blend of claim 1, wherein theratio of Product 2 to Product 1 is about 95:5.
 13. The amla seed blendof claim 1, wherein the ratio of Product 2 to Product 1 is about 3:1.14. The amla seed blend of claim 1, wherein the ratio of Product 2 toProduct 1 is about 1:5.
 15. The amla seed blend of claim 1, wherein theratio of Product 2 to Product 1 is about 1:10.
 16. The amla seed blendof claim 1, wherein the ratio of Product 2 to Product 1 is about 1:3.